Methods and compositions for the recombinant biosynthesis of n-alkanes

ABSTRACT

The present disclosure identifies methods and compositions for modifying photoautotrophic organisms as hosts, such that the organisms efficiently convert carbon dioxide and light into n-alkanes, and in particular the use of such organisms for the commercial production of n-alkanes and related molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to earlier filed U.S. ProvisionalPatent Application No. 61/224,463 filed, Jul. 9, 2009 and U.S.Provisional Patent Application No. 61/228,937, filed Jul. 27, 2009, thedisclosures of which are incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Apr. 13, 2010, is named16111US_SeqList.txt, lists 128 sequences, and is 336 kb in size.

FIELD OF THE INVENTION

The present disclosure relates to methods for conferringalkane-producing properties to a heterotrophic or photoautotrophic host,such that the modified host can be used in the commercial production ofbioalkanes.

BACKGROUND OF THE INVENTION

Many existing photoautotrophic organisms (i.e., plants, algae, andphotosynthetic bacteria) are poorly suited for industrial bioprocessingand have therefore not demonstrated commercial viability. Such organismstypically have slow doubling times (3-72 hrs) compared to industrializedheterotrophic organisms such as Escherichia coli (20 minutes),reflective of low total productivities. While a desire for the efficientbiosynthetic production of fuels has led to the development ofphotosynthetic microorganisms which produce alkyl esters of fatty acids,a need still exists for methods of producing hydrocarbons, e.g.,alkanes, using photosynthetic organisms.

SUMMARY OF THE INVENTION

The present invention provides, in certain embodiments, isolatedpolynucleotides comprising or consisting of nucleic acid sequencesselected from the group consisting of the coding sequences for AAR andADM enzymes, nucleic acid sequences that are codon-optimized variants ofthese sequences, and related nucleic acid sequences and fragments.

An AAR enzyme refers to an enzyme with the amino acid sequence of theSYNPCC7942_(—)1594 protein (SEQ ID NO: 6) or a homolog thereof, whereina SYNPCC7942_(—)1594 homolog is a protein whose BLAST alignment (i)covers >90% length of SYNPCC7942_(—)1594, (ii) covers >90% of the lengthof the matching protein, and (iii) has >50% identity withSYNPCC7942_(—)1594 (when optimally aligned using the parameters providedherein), and retains the functional activity of SYNPCC7942_(—)1594,i.e., the conversion of an acyl-ACP to an alkanal. An ADM enzyme refersto an enzyme with the amino acid sequence of the SYNPCC7942_(—)1593protein (SEQ ID NO: 8) or a homolog thereof, wherein aSYNPCC7942_(—)1593 homolog is defined as a protein whose amino acidsequence alignment (i) covers >90% length of SYNPCC7942_(—)1593, (ii)covers >90% of the length of the matching protein, and (iii) has >50%identity with SYNPCC7942_(—)1593 (when aligned using the preferredparameters provided herein), and retains the functional activity ofSYNPCC7942_(—)1593, i.e., the conversion of an n-alkanal to an(n−1)-alkane. Exemplary AAR and ADM enzymes are listed in Table 1 andTable 2, respectively. Genes encoding AAR or ADM enzymes are referred toherein as AAR genes or ADM genes, respectively.

Preferred parameters for BLASTp are: Expectation value: 10 (default);Filter: seg (default); Cost to open a gap: 11 (default); Cost to extenda gap: 1 (default); Max. alignments: 100 (default); Word size: 11(default); No. of descriptions: 100 (default); Penalty Matrix:BLOWSUM62.

While Applicants refer herein to a putative alkanal decarboxylativemonooxygenase activity, Applicants do so without intending to be boundto any particular reaction mechanism unless expressly set forth. Forexample, whether the enzyme encoded by SYNPCC7942_(—)1594 or any otherADM gene carries out a decarbonylase or a decarboxylase reaction doesnot affect the utility of Applicants' invention, unless expressly setforth herein to the contrary.

The present invention further provides isolated polypeptides comprisingor consisting of polypeptide sequences selected from the groupconsisting of the sequences listed in Table 1 and Table 2, and relatedpolypeptide sequences, fragments and fusions. Antibodies thatspecifically bind to the isolated polypeptides of the present inventionare also contemplated.

The present invention also provides methods for expressing aheterologous nucleic acid sequence encoding AAR and ADM in a host celllacking catalytic activity for AAR and ADM (thereby conferring n-alkaneproducing capability in the host cell), or for expressing a nucleic acidencoding AAR and ADM in a host cell which comprises native AAR and/orADM activity (thereby enhancing n-alkane producing capability in thehost cell).

In addition, the present invention provides methods for producingcarbon-based products of interest using the AAR and ADM genes, proteinsand host cells described herein. For example, in one embodiment theinvention provides a method for producing hydrocarbons, comprising: (i)culturing an engineered cyanobacterium in a culture medium, wherein saidengineered cyanobacterium comprises a recombinant AAR enzyme and arecombinant ADM enzyme; and (ii) exposing said engineered cyanobacteriumto light and carbon dioxide, wherein said exposure results in theconversion of said carbon dioxide by said engineered cynanobacteriuminto n-alkanes, wherein at least one of said n-alkanes is selected fromthe group consisting of n-tridecane, n-tetradecane, n-pentadecane,n-hexadecane, and n-heptadecane, and wherein the amount of saidn-alkanes produced is between 0.1% and 5% dry cell weight and at leasttwo times the amount produced by an otherwise identical cyanobacterium,cultured under identical conditions, but lacking said recombinant AARand ADM enzymes.

In a related embodiment, the amount on n-alkanes produced by theengineered cyanobacterium is at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, or 1%, and at least two times the amountproducted by an otherwise identical cyanobacterium, cultured underidentical conditions, but lacking said recombinant AAR and ADM enzymes.

In a related embodiment, at least one of said recombinant enzymes isheterologous with respect to said engineered cyanobacterium. In anotherembodiment, said cyanobacterium does not synthesize alkanes in theabsence of the expression of one or both of the recombinant enzymes. Inanother embodiment, at least one of said recombinant AAR or ADM enzymesis not heterologous to said engineered cyanobacterium.

In another related embodiment of the method, said engineeredcyanobacterium further produces at least one n-alkene or n-alkanol. Inyet another embodiment, the engineered cyanobacterium produces at leastone n-alkene or n-alkanol selected from the group consisting ofn-pentadecene, n-heptadecene, and 1-octadecanol. In a relatedembodiment, said n-alkanes comprise predominantly n-heptadecane,n-pentadecane or a combination thereof. In a related embodiment, moren-heptadecane and/or n-pentadecane are produced than all other n-alkaneproducts combined. In yet another related embodiment, more n-heptadecaneand/or n-pentadecane are produced by the engineered cyanobacterium thanany other n-alkane or n-alkene produced by the engineeredcyanobacterium. In yet another related embodiment, at least onen-pentadecene produced by said engineered cyanobacterium is selectedfrom the group consisting of cis-3-heptadecene and cis-4-pentadecene. Inyet another related embodiment, at least one n-heptadecene produced bysaid engineered cyanobacterium is selected from the group consisting ofcis-4-pentadecene, cis-6-heptadecene, cis-8-heptadecene,cis-9-heptadecene, and cis, cis-heptadec-di-ene.

In yet another related embodiment, the invention further provides a stepof isolating at least one n-alkane, n-alkene or n-alkanol from saidengineered cyanobacterium or said culture medium. In yet another relatedembodiment, the engineered cyanobacterium is cultured in a liquidmedium. In yet another related embodiment, the engineered cyanobacteriumis cultured in a photobioreactor.

In another related embodiment, the AAR and/or ADM enzymes are encoded bya plasmid. In yet another related embodiment, the AAR and/or ADM enzymesare encoded by recombinant genes incorporated into the genome of theengineered cyanobacterium. In yet another related embodiment, the AARand/or ADM enzymes are encoded by genes which are present in multiplecopies in said engineered cyanobacterium. In yet another relatedembodiment, the recombinant AAR and/or ADM enzymes are encoded by geneswhich are part of an operon, wherein the expression of said genes iscontrolled by a single promoter. In yet another related embodiment, therecombinant AAR and/or ADM enzymes are encoded by genes which areexpressed independently under the control of separate promoters. In yetanother related embodiment, expression of the recombinant AAR and/or ADMenzymes in an engineered cyanobacterium is controlled by a promoterselected from the group consisting of a cI promoter, a cpcB promoter, alacI-trc promoter, an EM7 promoter, and a aphII promoter.

In yet another related embodiment, the cyanobacterium species that isengineered to express recombinant AAR and/or ADM enzymes produces lessthan 0.1% DCW n-heptadecane or n-pentadecane in the absence of saidrecombinant AAR and/or ADM enzymes. In another related embodiment, theengineered cyanobacterium of the method is a thermophile. In yet anotherrelated embodiment, the engineered cyanobacterium of the method isselected from the group consisting of an engineered Synechococcus sp.PCC7002 and an engineered Thermosynechococcus elongatus BP-1.

In yet another related embodiment, the recombinant AAR and/or ADMenzymes are selected from the group of enzymes listed in Table 1 andTable 2, respectively. In yet another related embodiment, therecombinant AAR enzymes are selected from the group consisting ofSYNPCC7942_(—)1594, tll1312, and PMT9312_(—)0533. In yet another relatedembodiment, the recombinant ADM enzymes are selected from the groupconsisting of SYNPCC7942_(—)1593, tll1313, and PMT9312_(—)0532. In yetanother related embodiment, the recombinant AAR enzymes have the aminoacid sequences of SEQ ID NO:10 and SEQ ID NO:12, respectively. Incertain embodiments, the recombinant AAR and ADM enzymes are encoded bySEQ ID NOs: 9 and 11, respectively.

In yet another related embodiment, the method comprising culturing theengineered cyanobacterium in the presence of an antibiotic, wherein saidantibiotic selects for the presence of a recombinant gene encoding anAAR and/or ADM enzyme. In certain embodiments, the antibiotic isspectinomycin or kanamycin. In related embodiments, the amount ofspectinomycin in the culture media is between 100 and 700 μg/ml, e.g.,100, 200, 300, 400, 500, 600, or 700 μg/ml of spectinomycin can be addedto the culture media. In certain embodiments, the amount ofspectinomycin added is about 600 μg/ml, and the amount of n-alkanesproduced by the engineered cyanobacterium is at least about 3%, 4% or 5%DCW.

In another embodiment, the method for producing hydrocarbons comprisesculturing an cyanobacterium expressing recombinant AAR and/or ADMenzymes in the presence of an exogenous substrate for one or bothenzymes. In a related embodiment, the substrate is selected from thegroup consisting of an acyl-ACP, an acyl-CoA, a fatty aldehyde, a fattyalcohol, and a fatty ester.

In yet another embodiment, the invention provides a compositioncomprising an n-alkane produced by any of the recombinant biosyntheticmethods described herein. In yet another embodiment, the inventionprovides a composition comprising an n-alkene or n-alkanol produced byany of the recombinant biosynthetic methods described herein.

In certain embodiments, the invention provides an engineered host cellfor producing an n-alkane, wherein said cell comprises one or morerecombinant protein activities selected from the group consisting of anacyl-CoA reductase activity, an acyl-ACP reductase activity, an alkanaldecarboxylative monooxygenase activity, and an electron donor activity.In related embodiments, the host cell comprises a recombinant acyl-ACPreductase activity, a recombinant alkanal decarboxylative monooxygenaseactivity, and a recombinant electron donor activity. In otherembodiments, the host cell comprises a recombinant acyl-ACP reductaseactivity and a recombinant alkanal decarboxylative monooxygenaseactivity. In certain embodiments, the electron donor activity is aferredoxin. In certain related embodiments, the host cell is capable ofphotosynthesis. In still other related embodiments, the host cell is acyanobacterium. In still other embodiments, the host cell is agram-negative, a gram-positive bacteria, or a yeast cell.

In other embodiments, the invention provides an isolated or recombinantpolynucleotide comprising or consisting of a nucleic acid sequenceselected from the group consisting of: (a) SEQ ID NOs:1, 3, 5, 7, 9, 11,13 or 14; (b) a nucleic acid sequence that is a degenerate variant ofSEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 or 14; (c) a nucleic acid sequence atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%%, at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% orat least 99.9% identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 14; (d)a nucleic acid sequence that encodes a polypeptide having the amino acidsequence of SEQ ID NO:2, 4, 6, 8, 10 or 12; (e) a nucleic acid sequencethat encodes a polypeptide at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%%, at least 99.1%, at least 99.2%, at least99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%,at least 99.8% or at least 99.9% identical to SEQ ID NO:2, 4, 6, 8, 10,or 12; and (f) a nucleic acid sequence that hybridizes under stringentconditions to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 or 14. In relatedembodiments, the nucleic acid sequence encodes a polypeptide havingacyl-ACP reductase activity or alkanal decarboxylative monooxygenaseactivity.

In yet another embodiment, the invention provides an isolated, solublepolypeptide with alkanal decarboxylative monooxygenase activity wherein,in certain related embodiments, the polypeptide has an amino acidsequence of one of the proteins listed in Table 2. In relatedembodiments, the polypeptide has the amino acid sequence of SEQ ID NO:4, 8 or 12.

In yet another embodiment, the invention provides a method forsynthesizing an n-alkane from an acyl-ACP in vitro, comprising:contacting an acyl-ACP with a recombinant acyl-ACP reductase, whereinsaid acyl-ACP reductase converts said acyl-ACP to an n-alkanal; thencontacting said n-alkanal with a recombinant, soluble alkanaldecarboxylative monooxygenase in the presence of an electron donor,wherein said alkanal decarboxylative monooxygenase converts saidacyl-ACP to an n-alkane. In a related embodiment, the invention providesa method for synthesizing an n-alkane from an n-alkanal in vitro,comprising: contacting said n-alkanal with a recombinant, solublealkanal decarboxylative monooxygenase in the presence of an electrondonor, wherein said alkanal decarboxylative monooxygenase converts saidacyl-ACP to an n-alkane. In certain related embodiments, the electrondonor is a ferredoxin protein.

In another embodiment, the invention provides engineered cyanobacterialcells comprising recombinant AAR and ADM enzymes, wherein said cellscomprise between 0.1% and 5%, between 1% and 5%, or between 2% and 5%dry cell weight n-alkanes, wherein said n-alkanes are predominantlyn-pentadecane, n-heptadecane, or a combination thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts, in panel A, an enzymatic pathway for the production ofn-alkanes based on the sequential activity of (1) an AAR enzyme (e.g.,tll1312); and (2) an ADM enzyme (e.g., tll1313); B, Biosynthesis ofn-alkanal via acyl-CoA. Acyl-CoAs are typically intermediates of fattyacid degradation; C, Biosynthesis of n-alkanal via acyl-ACP. Acyl-ACP'sare typically intermediates of fatty acid biosynthesis. Note the threedifferent types of ACP reductase: (i) β-ketoacyl-ACP reductase, (ii)enoyl-ACP reductase, and (iii) acyl-ACP reductase. Acyl-ACP reductase, anew enzyme, generates the substrate for alkanal decarboxylativemonooxygenase. CoA, coenzyme A; ACP, acyl carrier protein; D, analternative acyl-CoA-mediated alkane biosynthetic pathway. Seeadditional discussion in Example 1, herein.

FIG. 2 represents 0-to-2700000-count total ion chromatograms of JCC9aand JCC1076 BHT (butylated hydroxytoluene)-acetone cell pellet extracts,as well as n-alkane and 1-alkanol authentic standards. Peaks assigned byMethod 1 are identified in regular font, those by Method 2 in italicfont.

FIG. 3 depicts MS fragmentation spectra of JCC1076 peaks assigned byMethod 1 (top mass spectrum of each panel), plotted against theirrespective NIST library hits (bottom mass spectrum of each panel). A,n-pentadecane; B, 1-tetradecanol; C, n-heptadecane; D, 1-hexadecanol.

FIG. 4A represents 0-to-7500000-count total ion chromatograms for theBHT-acetone extracts of JCC1113 and JCC1114 cell pellets, as well asC₁₃-C₂₀ n-alkane and C₁₄, C₁₆, and C₁₈ n-alkanol authentic standards; B,represents 0-to-720000-count total ion chromatograms for BHT-acetoneextracts of JCC1113 and JCC1114 cell pellets, as well as the n-alkaneand n-alkanol authentic standards mentioned in 4A.

FIG. 5 depicts MS fragmentation spectra of JCC1113 peaks assigned byMethod 1 (top mass spectrum of each panel), plotted against theirrespective NIST library hits (bottom mass spectrum of each panel). A,n-tridecane; B, n-tetradecane; C, n-pentadecane; D, n-hexadecane; E,n-heptadecane; F, 1-hexadecanol.

FIG. 6 represents 0-to-6100000-count total ion chromatograms of JCC1170and JCC1169 BHT-acetone cell pellet extracts versus those of the controlstrains JCC1113 and JCC1114. No hydrocarbon products were observed inJCC1169. The unidentified peak in JCC1170 is likely cis-11-octadecenal.

FIG. 7 depicts MS fragmentation spectra of JCC1170 peaks assigned byMethod 1 (top mass spectrum of each panel), plotted against theirrespective NIST library hits (bottom mass spectrum of each panel). A,1-tetradecanol; B, 1-hexadecanol.

FIG. 8A represents 0-to-75000000-count total ion chromatograms forBHT-acetone extracts of JCC1221, JCC1220, JCC1160b, JCC1160a, JCC1160and JCC879 cell pellets, as well as C₁₃-C₂₀ n-alkane and C₁₄, C₁₆, andC₁₈ n-alkanol authentic standards. The doublet around 18.0 minutescorresponds to nonadec-di-ene and 1-nonadecene, respectively (data notshown), n-alkenes that are naturally produced by JCC138; 8B represents0-to-2250000-count total ion chromatograms for BHT-acetone extracts ofJCC1221 and JCC879 cell pellets, as well as the n-alkane and n-alkanolauthentic standards mentioned in 8A.

FIG. 9 depicts MS fragmentation spectra of JCC1221 peaks assigned byMethod 1 (top mass spectrum of each panel), plotted against theirrespective NIST library hits (bottom mass spectrum of each panel). A,n-tridecane; B, n-tetradecane; C, n-pentadecane; D, n-hexadecane; E,n-heptadecane; F, 1-octadecanol.

FIG. 10 depicts intracellular n-alkane production as a function ofspectinomycin concentration in JCC1221.

FIG. 11 represents 0-to-1080000-count total ion chromatograms of theJCC1281 BHT-acetone cell pellet extractant versus that of the controlstrain JCC138, as well asof authentic standard n-alkanes.

FIG. 12 depicts MS fragmentation spectra of JCC1281 peaks assigned byMethod 1 (top mass spectrum of each panel), plotted against theirrespective NIST library hits (bottom mass spectrum of each panel). A,n-pentadecane; B, n-heptadecane.

FIG. 13 depicts MS fragmentation spectra of JCC3 peaks assigned byMethod 1 (top mass spectrum of each panel), plotted against theirrespective NIST library hits (bottom mass spectrum of each panel). A,n-pentadecane; B, n-hexadecane; C, n-heptadecane.

FIG. 14 depicts enhanced intracellular production of n-alkanes inJCC1084t compared to the control strain JCC1084. Error bars representstandard deviation of three independent observations.

FIG. 15 represents 0-to-31500000-count total ion chromatograms ofJCC1113 and JCC1221 BHT-acetone cell pellet extracts, as well asauthentic n-alkane standards.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall include theplural and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, biochemistry,enzymology, molecular and cellular biology, microbiology, genetics andprotein and nucleic acid chemistry and hybridization described hereinare those well known and commonly used in the art.

The methods and techniques of the present invention are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992, and Supplements to 2002); Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1990); Taylor and Drickamer,Introduction to Glycobiology, Oxford Univ. Press (2003); WorthingtonEnzyme Manual, Worthington Biochemical Corp., Freehold, N.J.; Handbookof Biochemistry: Section A Proteins, Vol I, CRC Press (1976); Handbookof Biochemistry: Section A Proteins, Vol II, CRC Press (1976);Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999).

All publications, patents and other references mentioned herein arehereby incorporated by reference in their entireties.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

The term “polynucleotide” or “nucleic acid molecule” refers to apolymeric form of nucleotides of at least 10 bases in length. The termincludes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNAmolecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA orRNA containing non-natural nucleotide analogs, non-nativeinternucleoside bonds, or both. The nucleic acid can be in anytopological conformation. For instance, the nucleic acid can besingle-stranded, double-stranded, triple-stranded, quadruplexed,partially double-stranded, branched, hairpinned, circular, or in apadlocked conformation.

Unless otherwise indicated, and as an example for all sequencesdescribed herein under the general format “SEQ ID NO:”, “nucleic acidcomprising SEQ ID NO:1” refers to a nucleic acid, at least a portion ofwhich has either (i) the sequence of SEQ ID NO:1, or (ii) a sequencecomplementary to SEQ ID NO:1. The choice between the two is dictated bythe context. For instance, if the nucleic acid is used as a probe, thechoice between the two is dictated by the requirement that the probe becomplementary to the desired target.

An “isolated” RNA, DNA or a mixed polymer is one which is substantiallyseparated from other cellular components that naturally accompany thenative polynucleotide in its natural host cell, e.g., ribosomes,polymerases and genomic sequences with which it is naturally associated.

As used herein, an “isolated” organic molecule (e.g., an alkane, alkene,or alkanal) is one which is substantially separated from the cellularcomponents (membrane lipids, chromosomes, proteins) of the host cellfrom which it originated, or from the medium in which the host cell wascultured. The term does not require that the biomolecule has beenseparated from all other chemicals, although certain isolatedbiomolecules may be purified to near homogeneity.

The term “recombinant” refers to a biomolecule, e.g., a gene or protein,that (1) has been removed from its naturally occurring environment, (2)is not associated with all or a portion of a polynucleotide in which thegene is found in nature, (3) is operatively linked to a polynucleotidewhich it is not linked to in nature, or (4) does not occur in nature.The term “recombinant” can be used in reference to cloned DNA isolates,chemically synthesized polynucleotide analogs, or polynucleotide analogsthat are biologically synthesized by heterologous systems, as well asproteins and/or mRNAs encoded by such nucleic acids.

As used herein, an endogenous nucleic acid sequence in the genome of anorganism (or the encoded protein product of that sequence) is deemed“recombinant” herein if a heterologous sequence is placed adjacent tothe endogenous nucleic acid sequence, such that the expression of thisendogenous nucleic acid sequence is altered. In this context, aheterologous sequence is a sequence that is not naturally adjacent tothe endogenous nucleic acid sequence, whether or not the heterologoussequence is itself endogenous (originating from the same host cell orprogeny thereof) or exogenous (originating from a different host cell orprogeny thereof). By way of example, a promoter sequence can besubstituted (e.g., by homologous recombination) for the native promoterof a gene in the genome of a host cell, such that this gene has analtered expression pattern. This gene would now become “recombinant”because it is separated from at least some of the sequences thatnaturally flank it.

A nucleic acid is also considered “recombinant” if it contains anymodifications that do not naturally occur to the corresponding nucleicacid in a genome. For instance, an endogenous coding sequence isconsidered “recombinant” if it contains an insertion, deletion or apoint mutation introduced artificially, e.g., by human intervention. A“recombinant nucleic acid” also includes a nucleic acid integrated intoa host cell chromosome at a heterologous site and a nucleic acidconstruct present as an episome.

As used herein, the phrase “degenerate variant” of a reference nucleicacid sequence encompasses nucleic acid sequences that can be translated,according to the standard genetic code, to provide an amino acidsequence identical to that translated from the reference nucleic acidsequence. The term “degenerate oligonucleotide” or “degenerate primer”is used to signify an oligonucleotide capable of hybridizing with targetnucleic acid sequences that are not necessarily identical in sequencebut that are homologous to one another within one or more particularsegments.

The term “percent sequence identity” or “identical” in the context ofnucleic acid sequences refers to the residues in the two sequences whichare the same when aligned for maximum correspondence. The length ofsequence identity comparison may be over a stretch of at least aboutnine nucleotides, usually at least about 20 nucleotides, more usually atleast about 24 nucleotides, typically at least about 28 nucleotides,more typically at least about 32 nucleotides, and preferably at leastabout 36 or more nucleotides. There are a number of different algorithmsknown in the art which can be used to measure nucleotide sequenceidentity. For instance, polynucleotide sequences can be compared usingFASTA, Gap or Bestfit, which are programs in Wisconsin Package Version10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences. Pearson, MethodsEnzymol. 183:63-98 (1990) (hereby incorporated by reference in itsentirety). For instance, percent sequence identity between nucleic acidsequences can be determined using FASTA with its default parameters (aword size of 6 and the NOPAM factor for the scoring matrix) or using Gapwith its default parameters as provided in GCG Version 6.1, hereinincorporated by reference. Alternatively, sequences can be comparedusing the computer program, BLAST (Altschul et al., J. Mol. Biol.215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993);Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res.7:649-656 (1997)), especially blastp or tblastn (Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997)).

The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 76%, 80%, 85%, preferablyat least about 90%, and more preferably at least about 95%, 96%, 97%,98% or 99% of the nucleotide bases, as measured by any well-knownalgorithm of sequence identity, such as FASTA, BLAST or Gap, asdiscussed above.

Alternatively, substantial homology or similarity exists when a nucleicacid or fragment thereof hybridizes to another nucleic acid, to a strandof another nucleic acid, or to the complementary strand thereof, understringent hybridization conditions. “Stringent hybridization conditions”and “stringent wash conditions” in the context of nucleic acidhybridization experiments depend upon a number of different physicalparameters. Nucleic acid hybridization will be affected by suchconditions as salt concentration, temperature, solvents, the basecomposition of the hybridizing species, length of the complementaryregions, and the number of nucleotide base mismatches between thehybridizing nucleic acids, as will be readily appreciated by thoseskilled in the art. One having ordinary skill in the art knows how tovary these parameters to achieve a particular stringency ofhybridization.

In general, “stringent hybridization” is performed at about 25° C. belowthe thermal melting point (T_(m)) for the specific DNA hybrid under aparticular set of conditions. “Stringent washing” is performed attemperatures about 5° C. lower than the T_(m) for the specific DNAhybrid under a particular set of conditions. The T_(m) is thetemperature at which 50% of the target sequence hybridizes to aperfectly matched probe. See Sambrook et al., Molecular Cloning: ALaboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989), page 9.51, hereby incorporated by reference.For purposes herein, “stringent conditions” are defined for solutionphase hybridization as aqueous hybridization (i.e., free of formamide)in 6×SSC (where 20×SSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1%SDS at 65° C. for 8-12 hours, followed by two washes in 0.2×SSC, 0.1%SDS at 65° C. for 20 minutes. It will be appreciated by the skilledworker that hybridization at 65° C. will occur at different ratesdepending on a number of factors including the length and percentidentity of the sequences which are hybridizing.

The nucleic acids (also referred to as polynucleotides) of this presentinvention may include both sense and antisense strands of RNA, cDNA,genomic DNA, and synthetic forms and mixed polymers of the above. Theymay be modified chemically or biochemically or may contain non-naturalor derivatized nucleotide bases, as will be readily appreciated by thoseof skill in the art. Such modifications include, for example, labels,methylation, substitution of one or more of the naturally occurringnucleotides with an analog, internucleotide modifications such asuncharged linkages (e.g., methyl phosphonates, phosphotriesters,phosphoramidates, carbamates, etc.), charged linkages (e.g.,phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g.,polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.) Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule. Other modifications can include, for example, analogs in whichthe ribose ring contains a bridging moiety or other structure such asthe modifications found in “locked” nucleic acids.

The term “mutated” when applied to nucleic acid sequences means thatnucleotides in a nucleic acid sequence may be inserted, deleted orchanged compared to a reference nucleic acid sequence. A singlealteration may be made at a locus (a point mutation) or multiplenucleotides may be inserted, deleted or changed at a single locus. Inaddition, one or more alterations may be made at any number of lociwithin a nucleic acid sequence. A nucleic acid sequence may be mutatedby any method known in the art including but not limited to mutagenesistechniques such as “error-prone PCR” (a process for performing PCR underconditions where the copying fidelity of the DNA polymerase is low, suchthat a high rate of point mutations is obtained along the entire lengthof the PCR product; see, e.g., Leung et al., Technique, 1:11-15 (1989)and Caldwell and Joyce, PCR Methods Applic. 2:28-33 (1992)); and“oligonucleotide-directed mutagenesis” (a process which enables thegeneration of site-specific mutations in any cloned DNA segment ofinterest; see, e.g., Reidhaar-Olson and Sauer, Science 241:53-57(1988)).

The term “attenuate” as used herein generally refers to a functionaldeletion, including a mutation, partial or complete deletion, insertion,or other variation made to a gene sequence or a sequence controlling thetranscription of a gene sequence, which reduces or inhibits productionof the gene product, or renders the gene product non-functional. In someinstances a functional deletion is described as a knockout mutation.Attenuation also includes amino acid sequence changes by altering thenucleic acid sequence, placing the gene under the control of a lessactive promoter, down-regulation, expressing interfering RNA, ribozymesor antisense sequences that target the gene of interest, or through anyother technique known in the art. In one example, the sensitivity of aparticular enzyme to feedback inhibition or inhibition caused by acomposition that is not a product or a reactant (non-pathway specificfeedback) is lessened such that the enzyme activity is not impacted bythe presence of a compound. In other instances, an enzyme that has beenaltered to be less active can be referred to as attenuated.

Deletion: The removal of one or more nucleotides from a nucleic acidmolecule or one or more amino acids from a protein, the regions oneither side being joined together.

Knock-out: A gene whose level of expression or activity has been reducedto zero. In some examples, a gene is knocked-out via deletion of some orall of its coding sequence. In other examples, a gene is knocked-out viaintroduction of one or more nucleotides into its open reading frame,which results in translation of a non-sense or otherwise non-functionalprotein product.

The term “vector” as used herein is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid,” which generally refersto a circular double stranded DNA loop into which additional DNAsegments may be ligated, but also includes linear double-strandedmolecules such as those resulting from amplification by the polymerasechain reaction (PCR) or from treatment of a circular plasmid with arestriction enzyme. Other vectors include cosmids, bacterial artificialchromosomes (BAC) and yeast artificial chromosomes (YAC). Another typeof vector is a viral vector, wherein additional DNA segments may beligated into the viral genome (discussed in more detail below). Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., vectors having an origin of replication whichfunctions in the host cell). Other vectors can be integrated into thegenome of a host cell upon introduction into the host cell, and arethereby replicated along with the host genome. Moreover, certainpreferred vectors are capable of directing the expression of genes towhich they are operatively linked. Such vectors are referred to hereinas “recombinant expression vectors” (or simply “expression vectors”).

“Operatively linked” or “operably linked” expression control sequencesrefers to a linkage in which the expression control sequence iscontiguous with the gene of interest to control the gene of interest, aswell as expression control sequences that act in trans or at a distanceto control the gene of interest.

The term “expression control sequence” as used herein refers topolynucleotide sequences which are necessary to affect the expression ofcoding sequences to which they are operatively linked. Expressioncontrol sequences are sequences which control the transcription,post-transcriptional events and translation of nucleic acid sequences.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g., ribosome binding sites); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence. The term “control sequences” is intended toinclude, at a minimum, all components whose presence is essential forexpression, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinant vectorhas been introduced. It should be understood that such terms areintended to refer not only to the particular subject cell but to theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. A recombinant host cell may be an isolated cell or cellline grown in culture or may be a cell which resides in a living tissueor organism.

The term “peptide” as used herein refers to a short polypeptide, e.g.,one that is typically less than about 50 amino acids long and moretypically less than about 30 amino acids long. The term as used hereinencompasses analogs and mimetics that mimic structural and thusbiological function.

The term “polypeptide” encompasses both naturally-occurring andnon-naturally-occurring proteins, and fragments, mutants, derivativesand analogs thereof. A polypeptide may be monomeric or polymeric.Further, a polypeptide may comprise a number of different domains eachof which has one or more distinct activities.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation (1) isnot associated with naturally associated components that accompany it inits native state, (2) exists in a purity not found in nature, wherepurity can be adjudged with respect to the presence of other cellularmaterial (e.g., is free of other proteins from the same species) (3) isexpressed by a cell from a different species, or (4) does not occur innature (e.g., it is a fragment of a polypeptide found in nature or itincludes amino acid analogs or derivatives not found in nature orlinkages other than standard peptide bonds). Thus, a polypeptide that ischemically synthesized or synthesized in a cellular system differentfrom the cell from which it naturally originates will be “isolated” fromits naturally associated components. A polypeptide or protein may alsobe rendered substantially free of naturally associated components byisolation, using protein purification techniques well known in the art.As thus defined, “isolated” does not necessarily require that theprotein, polypeptide, peptide or oligopeptide so described has beenphysically removed from its native environment.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has a deletion, e.g., an amino-terminal and/or carboxy-terminaldeletion compared to a full-length polypeptide. In a preferredembodiment, the polypeptide fragment is a contiguous sequence in whichthe amino acid sequence of the fragment is identical to thecorresponding positions in the naturally-occurring sequence. Fragmentstypically are at least 5, 6, 7, 8, 9 or 10 amino acids long, preferablyat least 12, 14, 16 or 18 amino acids long, more preferably at least 20amino acids long, more preferably at least 25, 30, 35, 40 or 45, aminoacids, even more preferably at least 50 or 60 amino acids long, and evenmore preferably at least 70 amino acids long.

A “modified derivative” refers to polypeptides or fragments thereof thatare substantially homologous in primary structural sequence but whichinclude, e.g., in vivo or in vitro chemical and biochemicalmodifications or which incorporate amino acids that are not found in thenative polypeptide. Such modifications include, for example,acetylation, carboxylation, phosphorylation, glycosylation,ubiquitination, labeling, e.g., with radionuclides, and variousenzymatic modifications, as will be readily appreciated by those skilledin the art. A variety of methods for labeling polypeptides and ofsubstituents or labels useful for such purposes are well known in theart, and include radioactive isotopes such as ¹²⁵I, ³²P, ³⁵S, and ³H,ligands which bind to labeled antiligands (e.g., antibodies),fluorophores, chemiluminescent agents, enzymes, and antiligands whichcan serve as specific binding pair members for a labeled ligand. Thechoice of label depends on the sensitivity required, ease of conjugationwith the primer, stability requirements, and available instrumentation.Methods for labeling polypeptides are well known in the art. See, e.g.,Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates (1992, and Supplements to 2002) (herebyincorporated by reference).

The term “fusion protein” refers to a polypeptide comprising apolypeptide or fragment coupled to heterologous amino acid sequences.Fusion proteins are useful because they can be constructed to containtwo or more desired functional elements from two or more differentproteins. A fusion protein comprises at least 10 contiguous amino acidsfrom a polypeptide of interest, more preferably at least 20 or 30 aminoacids, even more preferably at least 40, 50 or 60 amino acids, yet morepreferably at least 75, 100 or 125 amino acids. Fusions that include theentirety of the proteins of the present invention have particularutility. The heterologous polypeptide included within the fusion proteinof the present invention is at least 6 amino acids in length, often atleast 8 amino acids in length, and usefully at least 15, 20, and 25amino acids in length. Fusions that include larger polypeptides, such asan IgG Fc region, and even entire proteins, such as the greenfluorescent protein (“GFP”) chromophore-containing proteins, haveparticular utility. Fusion proteins can be produced recombinantly byconstructing a nucleic acid sequence which encodes the polypeptide or afragment thereof in frame with a nucleic acid sequence encoding adifferent protein or peptide and then expressing the fusion protein.Alternatively, a fusion protein can be produced chemically bycrosslinking the polypeptide or a fragment thereof to another protein.

As used herein, the term “antibody” refers to a polypeptide, at least aportion of which is encoded by at least one immunoglobulin gene, orfragment thereof, and that can bind specifically to a desired targetmolecule. The term includes naturally-occurring forms, as well asfragments and derivatives.

Fragments within the scope of the term “antibody” include those producedby digestion with various proteases, those produced by chemical cleavageand/or chemical dissociation and those produced recombinantly, so longas the fragment remains capable of specific binding to a targetmolecule. Among such fragments are Fab, Fab′, Fv, F(ab′).sub.2, andsingle chain Fv (scFv) fragments.

Derivatives within the scope of the term include antibodies (orfragments thereof) that have been modified in sequence, but remaincapable of specific binding to a target molecule, including:interspecies chimeric and humanized antibodies; antibody fusions;heteromeric antibody complexes and antibody fusions, such as diabodies(bispecific antibodies), single-chain diabodies, and intrabodies (see,e.g., Intracellular Antibodies: Research and Disease Applications,(Marasco, ed., Springer-Verlag New York, Inc., 1998), the disclosure ofwhich is incorporated herein by reference in its entirety).

As used herein, antibodies can be produced by any known technique,including harvest from cell culture of native B lymphocytes, harvestfrom culture of hybridomas, recombinant expression systems and phagedisplay.

The term “non-peptide analog” refers to a compound with properties thatare analogous to those of a reference polypeptide. A non-peptidecompound may also be termed a “peptide mimetic” or a “peptidomimetic.”See, e.g., Jones, Amino Acid and Peptide Synthesis, Oxford UniversityPress (1992); Jung, Combinatorial Peptide and Nonpeptide Libraries: AHandbook, John Wiley (1997); Bodanszky et al., Peptide Chemistry—APractical Textbook, Springer Verlag (1993); Synthetic Peptides: A UsersGuide, (Grant, ed., W. H. Freeman and Co., 1992); Evans et al., J. Med.Chem. 30:1229 (1987); Fauchere, J. Adv. Drug Res. 15:29 (1986); Veberand Freidinger, Trends Neurosci., 8:392-396 (1985); and references sitedin each of the above, which are incorporated herein by reference. Suchcompounds are often developed with the aid of computerized molecularmodeling. Peptide mimetics that are structurally similar to usefulpeptides of the present invention may be used to produce an equivalenteffect and are therefore envisioned to be part of the present invention.

A “polypeptide mutant” or “mutein” refers to a polypeptide whosesequence contains an insertion, duplication, deletion, rearrangement orsubstitution of one or more amino acids compared to the amino acidsequence of a native or wild-type protein. A mutein may have one or moreamino acid point substitutions, in which a single amino acid at aposition has been changed to another amino acid, one or more insertionsand/or deletions, in which one or more amino acids are inserted ordeleted, respectively, in the sequence of the naturally-occurringprotein, and/or truncations of the amino acid sequence at either or boththe amino or carboxy termini. A mutein may have the same but preferablyhas a different biological activity compared to the naturally-occurringprotein.

A mutein has at least 85% overall sequence homology to its wild-typecounterpart. Even more preferred are muteins having at least 90% overallsequence homology to the wild-type protein.

In an even more preferred embodiment, a mutein exhibits at least 95%sequence identity, even more preferably 98%, even more preferably 99%and even more preferably 99.9% overall sequence identity.

Sequence homology may be measured by any common sequence analysisalgorithm, such as Gap or Bestfit.

Amino acid substitutions can include those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinity or enzymatic activity, and (5) confer or modify otherphysicochemical or functional properties of such analogs.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2^(nd) ed.1991), which is incorporated herein by reference. Stereoisomers (e.g.,D-amino acids) of the twenty conventional amino acids, unnatural aminoacids such as α-, α-disubstituted amino acids, N-alkyl amino acids, andother unconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4-hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand end corresponds to the amino terminal end and the right-handend corresponds to the carboxy-terminal end, in accordance with standardusage and convention.

A protein has “homology” or is “homologous” to a second protein if thenucleic acid sequence that encodes the protein has a similar sequence tothe nucleic acid sequence that encodes the second protein.Alternatively, a protein has homology to a second protein if the twoproteins have “similar” amino acid sequences. (Thus, the term“homologous proteins” is defined to mean that the two proteins havesimilar amino acid sequences.) As used herein, homology between tworegions of amino acid sequence (especially with respect to predictedstructural similarities) is interpreted as implying similarity infunction.

When “homologous” is used in reference to proteins or peptides, it isrecognized that residue positions that are not identical often differ byconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of homology may be adjusted upwardsto correct for the conservative nature of the substitution. Means formaking this adjustment are well known to those of skill in the art. See,e.g., Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89 (hereinincorporated by reference).

The following six groups each contain amino acids that are conservativesubstitutions for one another: 1) Serine (S), Threonine (T); 2) AsparticAcid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine(M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W).

Sequence homology for polypeptides, which is also referred to as percentsequence identity, is typically measured using sequence analysissoftware. See, e.g., the Sequence Analysis Software Package of theGenetics Computer Group (GCG), University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using a measure of homology assignedto various substitutions, deletions and other modifications, includingconservative amino acid substitutions. For instance, GCG containsprograms such as “Gap” and “Bestfit” which can be used with defaultparameters to determine sequence homology or sequence identity betweenclosely related polypeptides, such as homologous polypeptides fromdifferent species of organisms or between a wild-type protein and amutein thereof. See, e.g., GCG Version 6.1.

A preferred algorithm when comparing a particular polypeptide sequenceto a database containing a large number of sequences from differentorganisms is the computer program BLAST (Altschul et al., J. Mol. Biol.215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993);Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res.7:649-656 (1997)), especially blastp or tblastn (Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997)).

Preferred parameters for BLASTp are: Expectation value: 10 (default);Filter: seg (default); Cost to open a gap: 11 (default); Cost to extenda gap: 1 (default); Max. alignments: 100 (default); Word size: 11(default); No. of descriptions: 100 (default); Penalty Matrix:BLOWSUM62.

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acid residues, usually at least about 20residues, more usually at least about 24 residues, typically at leastabout 28 residues, and preferably more than about 35 residues. Whensearching a database containing sequences from a large number ofdifferent organisms, it is preferable to compare amino acid sequences.Database searching using amino acid sequences can be measured byalgorithms other than blastp known in the art. For instance, polypeptidesequences can be compared using FASTA, a program in GCG Version 6.1.FASTA provides alignments and percent sequence identity of the regionsof the best overlap between the query and search sequences. Pearson,Methods Enzymol. 183:63-98 (1990) (incorporated by reference herein).For example, percent sequence identity between amino acid sequences canbe determined using FASTA with its default parameters (a word size of 2and the PAM250 scoring matrix), as provided in GCG Version 6.1, hereinincorporated by reference.

“Specific binding” refers to the ability of two molecules to bind toeach other in preference to binding to other molecules in theenvironment. Typically, “specific binding” discriminates overadventitious binding in a reaction by at least two-fold, more typicallyby at least 10-fold, often at least 100-fold. Typically, the affinity oravidity of a specific binding reaction, as quantified by a dissociationconstant, is about 10⁻⁷ M or stronger (e.g., about 10⁻⁸ M, 10⁻⁹ M oreven stronger).

“Percent dry cell weight” refers to a measurement of hydrocarbonproduction obtained as follows: a defined volume of culture iscentrifuged to pellet the cells. Cells are washed then dewetted by atleast one cycle of microcentrifugation and aspiration. Cell pellets arelyophilized overnight, and the tube containing the dry cell mass isweighed again such that the mass of the cell pellet can be calculatedwithin ±0.1 mg. At the same time cells are processed for dry cell weightdetermination, a second sample of the culture in question is harvested,washed, and dewetted. The resulting cell pellet, corresponding to 1-3 mgof dry cell weight, is then extracted by vortexing in approximately 1 mlacetone plus butylated hydroxytolune (BHT) as antioxidant and aninternal standard, e.g., n-heptacosane. Cell debris is then pelleted bycentrifugation and the supernatant (extractant) is taken for analysis byGC. For accurate quantitation of n-alkanes, flame ionization detection(FID) was used as opposed to MS total ion count. n-Alkane concentrationsin the biological extracts were calculated using calibrationrelationships between GC-FID peak area and known concentrations ofauthentic n-alkane standards. Knowing the volume of the extractant, theresulting concentrations of the n-alkane species in the extracant, andthe dry cell weight of the cell pellet extracted, the percentage of drycell weight that comprised n-alkanes can be determined.

The term “region” as used herein refers to a physically contiguousportion of the primary structure of a biomolecule. In the case ofproteins, a region is defined by a contiguous portion of the amino acidsequence of that protein.

The term “domain” as used herein refers to a structure of a biomoleculethat contributes to a known or suspected function of the biomolecule.Domains may be co-extensive with regions or portions thereof; domainsmay also include distinct, non-contiguous regions of a biomolecule.Examples of protein domains include, but are not limited to, an Igdomain, an extracellular domain, a transmembrane domain, and acytoplasmic domain.

As used herein, the term “molecule” means any compound, including, butnot limited to, a small molecule, peptide, protein, sugar, nucleotide,nucleic acid, lipid, etc., and such a compound can be natural orsynthetic.

“Carbon-based Products of Interest” include alcohols such as ethanol,propanol, isopropanol, butanol, fatty alcohols, fatty acid esters, waxesters; hydrocarbons and alkanes such as propane, octane, diesel, JetPropellant 8 (JP8); polymers such as terephthalate, 1,3-propanediol,1,4-butanediol, polyols, Polyhydroxyalkanoates (PHA),poly-beta-hydroxybutyrate (PHB), acrylate, adipic acid, ε-caprolactone,isoprene, caprolactam, rubber; commodity chemicals such as lactate,Docosahexaenoic acid (DHA), 3-hydroxypropionate, γ-valerolactone,lysine, serine, aspartate, aspartic acid, sorbitol, ascorbate, ascorbicacid, isopentenol, lanosterol, omega-3 DHA, lycopene, itaconate,1,3-butadiene, ethylene, propylene, succinate, citrate, citric acid,glutamate, malate, 3-hydroxypropionic acid (HPA), lactic acid, THF,gamma butyrolactone, pyrrolidones, hydroxybutyrate, glutamic acid,levulinic acid, acrylic acid, malonic acid; specialty chemicals such ascarotenoids, isoprenoids, itaconic acid; pharmaceuticals andpharmaceutical intermediates such as 7-aminodeacetoxycephalosporanicacid (7-ADCA)/cephalosporin, erythromycin, polyketides, statins,paclitaxel, docetaxel, terpenes, peptides, steroids, omega fatty acidsand other such suitable products of interest. Such products are usefulin the context of biofuels, industrial and specialty chemicals, asintermediates used to make additional products, such as nutritionalsupplements, neutraceuticals, polymers, paraffin replacements, personalcare products and pharmaceuticals.

Biofuel: A biofuel refers to any fuel that derives from a biologicalsource. Biofuel can refer to one or more hydrocarbons, one or morealcohols, one or more fatty esters or a mixture thereof.

Hydrocarbon: The term generally refers to a chemical compound thatconsists of the elements carbon (C), hydrogen (H) and optionally oxygen(O). There are essentially three types of hydrocarbons, e.g., aromatichydrocarbons, saturated hydrocarbons and unsaturated hydrocarbons suchas alkenes, alkynes, and dienes. The term also includes fuels, biofuels,plastics, waxes, solvents and oils. Hydrocarbons encompass biofuels, aswell as plastics, waxes, solvents and oils.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this present invention pertains. Exemplary methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ofthe present invention and will be apparent to those of skill in the art.All publications and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control. The materials,methods, and examples are illustrative only and not intended to belimiting.

Throughout this specification and claims, the word “comprise” orvariations such as “comprises” or “comprising”, will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

Nucleic Acid Sequences

Alkanes, also known as paraffins, are chemical compounds that consistonly of the elements carbon (C) and hydrogen (H) (i.e., hydrocarbons),wherein these atoms are linked together exclusively by single bonds(i.e., they are saturated compounds) without any cyclic structure.n-Alkanes are linear, i.e., unbranched, alkanes. Together, AAR and ADMenzymes function to synthesize n-alkanes from acyl-ACP molecules.

Accordingly, the present invention provides isolated nucleic acidmolecules for genes encoding AAR and ADM enzymes, and variants thereof.Exemplary full-length nucleic acid sequences for genes encoding AAR arepresented as SEQ ID NOs: 1, 5, and 13, and the corresponding amino acidsequences are presented as SEQ ID NOs: 2, 6, and 10, respectively.Exemplary full-length nucleic acid sequences for genes encoding ADM arepresented as SEQ ID NOs: 3, 7, 14, and the corresponding amino acidsequences are presented as SEQ ID NOs: 4, 8, and 12, respectively.Additional nucleic acids provided by the invention include any of thegenes encoding the AAR and ADM enzymes in Table 1 and Table 2,respectively.

In one embodiment, the present invention provides an isolated nucleicacid molecule having a nucleic acid sequence comprising or consisting ofa gene coding for AAR and ADM, and homologs, variants and derivativesthereof expressed in a host cell of interest. The present invention alsoprovides a nucleic acid molecule comprising or consisting of a sequencewhich is a codon-optimized version of the AAR and ADM genes describedherein (e.g., SEQ ID NO: 9 and SEQ ID NO: 11, which are optimized forthe expression of the AAR and ADM genes of Prochlorococcus marinus MIT9312 in Synechoccocus sp. PCC 7002). In a further embodiment, thepresent invention provides a nucleic acid molecule and homologs,variants and derivatives of the molecule comprising or consisting of asequence which is a variant of the AAR or ADM gene having at least 76%identity to the wild-type gene. The nucleic acid sequence can bepreferably 80%, 85%, 90%, 95%, 98%, 99%, 99.9% or even higher identityto the wild-type gene.

In another embodiment, the nucleic acid molecule of the presentinvention encodes a polypeptide having the amino acid sequence of SEQ IDNO:2, 4, 6, 8, 10 or 12. Preferably, the nucleic acid molecule of thepresent invention encodes a polypeptide sequence of at least 50%, 60,70%, 80%, 85%, 90% or 95% identity to SEQ ID NO:2, 4, 6, 8, 10 or 12 andthe identity can even more preferably be 96%, 97%, 98%, 99%, 99.9% oreven higher.

The present invention also provides nucleic acid molecules thathybridize under stringent conditions to the above-described nucleic acidmolecules. As defined above, and as is well known in the art, stringenthybridizations are performed at about 25° C. below the thermal meltingpoint (T_(m)) for the specific DNA hybrid under a particular set ofconditions, where the T_(m) is the temperature at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Stringentwashing is performed at temperatures about 5° C. lower than the T_(m)for the specific DNA hybrid under a particular set of conditions.

Nucleic acid molecules comprising a fragment of any one of theabove-described nucleic acid sequences are also provided. Thesefragments preferably contain at least 20 contiguous nucleotides. Morepreferably the fragments of the nucleic acid sequences contain at least25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or even more contiguousnucleotides.

The nucleic acid sequence fragments of the present invention displayutility in a variety of systems and methods. For example, the fragmentsmay be used as probes in various hybridization techniques. Depending onthe method, the target nucleic acid sequences may be either DNA or RNA.The target nucleic acid sequences may be fractionated (e.g., by gelelectrophoresis) prior to the hybridization, or the hybridization may beperformed on samples in situ. One of skill in the art will appreciatethat nucleic acid probes of known sequence find utility in determiningchromosomal structure (e.g., by Southern blotting) and in measuring geneexpression (e.g., by Northern blotting). In such experiments, thesequence fragments are preferably detectably labeled, so that theirspecific hydridization to target sequences can be detected andoptionally quantified. One of skill in the art will appreciate that thenucleic acid fragments of the present invention may be used in a widevariety of blotting techniques not specifically described herein.

It should also be appreciated that the nucleic acid sequence fragmentsdisclosed herein also find utility as probes when immobilized onmicroarrays. Methods for creating microarrays by deposition and fixationof nucleic acids onto support substrates are well known in the art.Reviewed in DNA Microarrays: A Practical Approach (Practical ApproachSeries), Schena (ed.), Oxford University Press (1999) (ISBN:0199637768); Nature Genet. 21(1)(suppl):1-60 (1999); Microarray Biochip:Tools and Technology, Schena (ed.), Eaton PublishingCompany/BioTechniques Books Division (2000) (ISBN: 1881299376), thedisclosures of which are incorporated herein by reference in theirentireties. Analysis of, for example, gene expression using microarrayscomprising nucleic acid sequence fragments, such as the nucleic acidsequence fragments disclosed herein, is a well-established utility forsequence fragments in the field of cell and molecular biology. Otheruses for sequence fragments immobilized on microarrays are described inGerhold et al., Trends Biochem. Sci. 24:168-173 (1999) and Zweiger,Trends Biotechnol. 17:429-436 (1999); DNA Microarrays: A PracticalApproach (Practical Approach Series), Schena (ed.), Oxford UniversityPress (1999) (ISBN: 0199637768); Nature Genet. 21(1)(suppl):1-60 (1999);Microarray Biochip: Tools and Technology, Schena (ed.), Eaton PublishingCompany/BioTechniques Books Division (2000) (ISBN: 1881299376), thedisclosure of each of which is incorporated herein by reference in itsentirety.

As is well known in the art, enzyme activities can be measured invarious ways. For example, the pyrophosphorolysis of OMP may be followedspectroscopically (Grubmeyer et al., (1993) J. Biol. Chem.268:20299-20304). Alternatively, the activity of the enzyme can befollowed using chromatographic techniques, such as by high performanceliquid chromatography (Chung and Sloan, (1986) J. Chromatogr.371:71-81). As another alternative the activity can be indirectlymeasured by determining the levels of product made from the enzymeactivity. These levels can be measured with techniques including aqueouschloroform/methanol extraction as known and described in the art (Cf. M.Kates (1986) Techniques of Lipidology; Isolation, analysis andidentification of Lipids. Elsevier Science Publishers, New York (ISBN:0444807322)). More modern techniques include using gas chromatographylinked to mass spectrometry (Niessen, W. M. A. (2001). Current practiceof gas chromatography—mass spectrometry. New York, N.Y.: Marcel Dekker.(ISBN: 0824704738)). Additional modern techniques for identification ofrecombinant protein activity and products including liquidchromatography-mass spectrometry (LCMS), high performance liquidchromatography (HPLC), capillary electrophoresis, Matrix-Assisted LaserDesorption Ionization time of flight-mass spectrometry (MALDI-TOF MS),nuclear magnetic resonance (NMR), near-infrared (NIR) spectroscopy,viscometry (Knothe, G (1997) Am. Chem. Soc. Symp. Series, 666: 172-208),titration for determining free fatty acids (Komers (1997) Fett/Lipid,99(2): 52-54), enzymatic methods (Bailer (1991) Fresenius J. Anal. Chem.340(3): 186), physical property-based methods, wet chemical methods,etc. can be used to analyze the levels and the identity of the productproduced by the organisms of the present invention. Other methods andtechniques may also be suitable for the measurement of enzyme activity,as would be known by one of skill in the art.

Vectors

Also provided are vectors, including expression vectors, which comprisethe above nucleic acid molecules of the present invention, as describedfurther herein. In a first embodiment, the vectors include the isolatednucleic acid molecules described above. In an alternative embodiment,the vectors of the present invention include the above-described nucleicacid molecules operably linked to one or more expression controlsequences. The vectors of the instant invention may thus be used toexpress an AAR and/or ADM polypeptide contributing to n-alkane producingactivity by a host cell.

Vectors useful for expression of nucleic acids in prokaryotes are wellknown in the art.

Isolated Polypeptides

According to another aspect of the present invention, isolatedpolypeptides (including muteins, allelic variants, fragments,derivatives, and analogs) encoded by the nucleic acid molecules of thepresent invention are provided. In one embodiment, the isolatedpolypeptide comprises the polypeptide sequence corresponding to SEQ IDNO:2, 4, 6, 8 10 or 12. In an alternative embodiment of the presentinvention, the isolated polypeptide comprises a polypeptide sequence atleast 85% identical to SEQ ID NO:2, 4, 6, 8, 10 or 12. Preferably theisolated polypeptide of the present invention has at least 50%, 60, 70%,80%, 85%, 90%, 95%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%,98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, 99.9% or even higher identity to SEQ ID NO:2, 4, 6, 8, 10or 12.

According to other embodiments of the present invention, isolatedpolypeptides comprising a fragment of the above-described polypeptidesequences are provided. These fragments preferably include at least 20contiguous amino acids, more preferably at least 25, 30, 35, 40, 45, 50,60, 70, 80, 90, 100 or even more contiguous amino acids.

The polypeptides of the present invention also include fusions betweenthe above-described polypeptide sequences and heterologous polypeptides.The heterologous sequences can, for example, include sequences designedto facilitate purification, e.g. histidine tags, and/or visualization ofrecombinantly-expressed proteins. Other non-limiting examples of proteinfusions include those that permit display of the encoded protein on thesurface of a phage or a cell, fusions to intrinsically fluorescentproteins, such as green fluorescent protein (GFP), and fusions to theIgG Fc region.

Host Cell Transformants

In another aspect of the present invention, host cells transformed withthe nucleic acid molecules or vectors of the present invention, anddescendants thereof, are provided. In some embodiments of the presentinvention, these cells carry the nucleic acid sequences of the presentinvention on vectors, which may but need not be freely replicatingvectors. In other embodiments of the present invention, the nucleicacids have been integrated into the genome of the host cells.

In a preferred embodiment, the host cell comprises one or more AAR orADM encoding nucleic acids which express AAR or ADM in the host cell.

In an alternative embodiment, the host cells of the present inventioncan be mutated by recombination with a disruption, deletion or mutationof the isolated nucleic acid of the present invention so that theactivity of the AAR and/or ADM protein(s) in the host cell is reduced oreliminated compared to a host cell lacking the mutation.

Selected or Engineered Microorganisms for the Production of Carbon-BasedProducts of Interest

Microorganism: Includes prokaryotic and eukaryotic microbial speciesfrom the Domains Archaea, Bacteria and Eucarya, the latter includingyeast and filamentous fungi, protozoa, algae, or higher Protista. Theterms “microbial cells” and “microbes” are used interchangeably with theterm microorganism.

A variety of host organisms can be transformed to produce a product ofinterest. Photoautotrophic organisms include eukaryotic plants andalgae, as well as prokaryotic cyanobacteria, green-sulfur bacteria,green non-sulfur bacteria, purple sulfur bacteria, and purple non-sulfurbacteria.

Extremophiles are also contemplated as suitable organisms. Suchorganisms withstand various environmental parameters such astemperature, radiation, pressure, gravity, vacuum, desiccation,salinity, pH, oxygen tension, and chemicals. They includehyperthermophiles, which grow at or above 80° C. such as Pyrolobusfumarii; thermophiles, which grow between 60-80° C. such asSynechococcus lividis; mesophiles, which grow between 15-60° C. andpsychrophiles, which grow at or below 15° C. such as Psychrobacter andsome insects. Radiation tolerant organisms include Deinococcusradiodurans. Pressure-tolerant organisms include piezophiles, whichtolerate pressure of 130 MPa. Weight-tolerant organisms includebarophiles. Hypergravity (e.g., >1 g) hypogravity (e.g., <1 g) tolerantorganisms are also contemplated. Vacuum tolerant organisms includetardigrades, insects, microbes and seeds. Dessicant tolerant andanhydrobiotic organisms include xerophiles such as Artemia salina;nematodes, microbes, fungi and lichens. Salt-tolerant organisms includehalophiles (e.g., 2-5 M NaCl) Halobacteriacea and Dunaliella salina.pH-tolerant organisms include alkaliphiles such as Natronobacterium,Bacillus firmus OF4, Spirulina spp. (e.g., pH>9) and acidophiles such asCyanidium caldarium, Ferroplasma sp. (e.g., low pH). Anaerobes, whichcannot tolerate O₂ such as Methanococcus jannaschii; microaerophils,which tolerate some O₂ such as Clostridium and aerobes, which require O₂are also contemplated. Gas-tolerant organisms, which tolerate pure CO₂include Cyanidium caldarium and metal tolerant organisms includemetalotolerants such as Ferroplasma acidarmanus (e.g., Cu, As, Cd, Zn),Ralstonia sp. CH34 (e.g., Zn, Co, Cd, Hg, Pb). Gross, Michael. Life onthe Edge: Amazing Creatures Thriving in Extreme Environments. New YorK:Plenum (1998) and Seckbach, J. “Search for Life in the Universe withTerrestrial Microbes Which Thrive Under Extreme Conditions.” InCristiano Batalli Cosmovici, Stuart Bowyer, and Dan Wertheimer, eds.,Astronomical and Biochemical Origins and the Search for Life in theUniverse, p. 511. Milan: Editrice Compositori (1997).

Plants include but are not limited to the following genera: Arabidopsis,Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus,Saccharum, Salix, Simmondsia and Zea.

Algae and cyanobacteria include but are not limited to the followinggenera: Acanthoceras, Acanthococcus, Acaryochloris, Achnanthes,Achnanthidium, Actinastrum, Actinochloris, Actinocyclus, Actinotaenium,Amphichrysis, Amphidinium, Amphikrikos, Amphipleura, Amphiprora,Amphithrix, Amphora, Anabaena, Anabaenopsis, Aneumastus, Ankistrodesmus,Ankyra, Anomoeoneis, Apatococcus, Aphanizomenon, Aphanocapsa,Aphanochaete, Aphanothece, Apiocystis, Apistonema, Arthrodesmus,Artherospira, Ascochloris, Asterionella, Asterococcus, Audouinella,Aulacoseira, Bacillaria, Balbiania, Bambusina, Bangia, Basichlamys,Batrachospermum, Binuclearia, Bitrichia, Blidingia, Botrdiopsis,Botrydium, Botryococcus, Botryosphaerella, Brachiomonas, Brachysira,Brachytrichia, Brebissonia, Bulbochaete, Bumilleria, Bumilleriopsis,Caloneis, Calothrix, Campylodiscus, Capsosiphon, Carteria, Catena,Cavinula, Centritractus, Centronella, Ceratium, Chaetoceros,Chaetochloris, Chaetomorpha, Chaetonella, Chaetonema, Chaetopeltis,Chaetophora, Chaetosphaeridium, Chamaesiphon, Chara, Characiochloris,Characiopsis, Characium, Charales, Chilomonas, Chlainomonas,Chlamydoblepharis, Chlamydocapsa, Chlamydomonas, Chlamydomonopsis,Chlamydomyxa, Chlamydonephris, Chlorangiella, Chlorangiopsis, Chlorella,Chlorobotrys, Chlorobrachis, Chlorochytrium, Chlorococcum, Chlorogloea,Chlorogloeopsis, Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema,Chlorophyta, Chlorosaccus, Chlorosarcina, Choricystis, Chromophyton,Chromulina, Chroococcidiopsis, Chroococcus, Chroodactylon, Chroomonas,Chroothece, Chrysamoeba, Chrysapsis, Chrysidiastrum, Chrysocapsa,Chrysocapsella, Chrysochaete, Chrysochromulina, Chrysococcus,Chrysocrinus, Chrysolepidomonas, Chrysolykos, Chrysonebula, Chrysophyta,Chrysopyxis, Chrysosaccus, Chrysophaerella, Chrysostephanosphaera,Clodophora, Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis,Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus,Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis,Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium,Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium,Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta,Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta,Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella,Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca,Cymatopleura, Cymbella, Cymbellonitzschia, Cystodinium Dactylococcopsis,Debarya, Denticula, Dermatochrysis, Dermocarpa, Dermocarpella,Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon,Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula,Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus,Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia, Dilabifilum,Dimorphococcus, Dinobryon, Dinococcus, Diplochloris, Diploneis,Diplostauron, Distrionella, Docidium, Draparnaldia, Dunaliella,Dysmorphococcus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema,Enteromorpha, Entocladia, Entomoneis, Entophysalis, Epichrysis,Epipyxis, Epithemia, Eremosphaera, Euastropsis, Euastrum, Eucapsis,Eucocconeis, Eudorina, Euglena, Euglenophyta, Eunotia, Eustigmatophyta,Eutreptia, Fallacia, Fischerella, Fragilaria, Fragilariforma, Franceia,Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis, Glaucophyta,Glenodiniopsis, Glenodinium, Gloeocapsa, Gloeochaete, Gloeochrysis,Gloeococcus, Gloeocystis, Gloeodendron, Gloeomonas, Gloeoplax,Gloeothece, Gloeotila, Gloeotrichia, Gloiodictyon, Golenkinia,Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema, Gomphosphaeria,Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum,Granulochloris, Granulocystopsis, Groenbladia, Gymnodinium, Gymnozyga,Gyrosigma, Haematococcus, Hafniomonas, Hallassia, Hammatoidea, Hannaea,Hantzschia, Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinium,Hemitoma, Heribaudiella, Heteromastix, Heterothrix, Hibberdia,Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema,Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium,Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum, Hydrocoryne,Hydrodictyon, Hydrosera, Hydrurus, Hyella, Hymenomonas, Isthmochloron,Johannesbaptistia, Juranyiella, Karayevia, Kathablepharis, Katodinium,Kephyrion, Keratococcus, Kirchneriella, Klebsormidium, Kolbesia,Koliella, Komarekia, Korshikoviella, Kraskella, Lagerheimia, Lagynion,Lamprothamnium, Lemanea, Lepocinclis, Leptosira, Lobococcus, Lobocystis,Lobomonas, Luticola, Lyngbya, Malleochloris, Mallomonas, Mantoniella,Marssoniella, Martyana, Mastigocoleus, Gastogloia, Melosira,Merismopedia, Mesostigma, Mesotaenium, Micractinium, Micrasterias,Microchaete, Microcoleus, Microcystis, Microglena, Micromonas,Microspora, Microthamnion, Mischococcus, Monochrysis, Monodus,Monomastix, Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis,Myochloris, Myromecia, Myxosarcina, Naegeliella, Nannochloris,Nautococcus, Navicula, Neglectella, Neidium, Nephroclamys, Nephrocytium,Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia,Nodularia, Nostoc, Ochromonas, Oedogonium, Oligochaetophora, Onychonema,Oocardium, Oocystis, Opephora, Ophiocytium, Orthoseira, Oscillatoria,Oxyneis, Pachycladella, Palmella, Palmodictyon, Pnadorina, Pannus,Paralia, Pascherina, Paulschulzia, Pediastrum, Pedinella, Pedinomonas,Pedinopera, Pelagodictyon, Penium, Peranema, Peridiniopsis, Peridinium,Peronia, Petroneis, Phacotus, Phacus, Phaeaster, Phaeodermatium,Phaeophyta, Phaeosphaera, Phaeothamnion, Phormidium, Phycopeltis,Phyllariochloris, Phyllocardium, Phyllomitas, Pinnularia, Pitophora,Placoneis, Planctonema, Planktosphaeria, Planothidium, Plectonema,Pleodorina, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus,Pleurosigma, Pleurosira, Pleurotaenium, Pocillomonas, Podohedra,Polyblepharides, Polychaetophora, Polyedriella, Polyedriopsis,Polygoniochloris, Polyepidomonas, Polytaenia, Polytoma, Polytomella,Porphyridium, Posteriochromonas, Prasinochloris, Prasinocladus,Prasinophyta, Prasiola, Prochlorphyta, Prochlorothrix, Protoderma,Protosiphon, Provasoliella, Prymnesium, Psammodictyon, Psammothidium,Pseudanabaena, Pseudenoclonium, Psuedocarteria, Pseudochate,Pseudocharacium, Pseudococcomyxa, Pseudodictyosphaerium,Pseudokephyrion, Pseudoncobyrsa, Pseudoquadrigula, Pseudosphaerocystis,Pseudostaurastrum, Pseudostaurosira, Pseudotetrastrum, Pteromonas,Punctastruata, Pyramichlamys, Pyramimonas, Pyrrophyta, Quadrichloris,Quadricoccus, Quadrigula, Radiococcus, Radiofilum, Raphidiopsis,Raphidocelis, Raphidonema, Raphidophyta, Peimeria, Rhabdoderma,Rhabdomonas, Rhizoclonium, Rhodomonas, Rhodophyta, Rhoicosphenia,Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus,Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix,Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia,Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis,Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium,Sirogonium, Skeletonema, Sorastrum, Spennatozopsis, Sphaerellocystis,Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma,Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum,Spondylosium, Sporotetras, Spumella, Staurastrum, Stauerodesmus,Stauroneis, Staurosira, Staurosirella, Stenopterobia, Stephanocostis,Stephanodiscus, Stephanoporos, Stephanosphaera, Stichococcus,Stichogloea, Stigeoclonium, Stigonema, Stipitococcus, Stokesiella,Strombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridium,Surirella, Sykidion, Symploca, Synechococcus, Synechocystis, Synedra,Synochromonas, Synura, Tabellaria, Tabularia, Teilingia, Temnogametum,Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus, Tetraedriella,Tetraedron, Tetraselmis, Tetraspora, Tetrastrum, Thalassiosira,Thamniochaete, Thorakochloris, Thorea, Tolypella, Tolypothrix,Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria,Tribonema, Trichodesmium, Trichodiscus, Trochiscia, Tryblionella,Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva, Vacuolaria,Vaucheria, Volvox, Volvulina, Westella, Woloszynskia, Xanthidium,Xanthophyta, Xenococcus, Zygnema, Zygnemopsis, and Zygonium. A partiallist of cyanobacteria that can be engineered to express recombinant AARand ADM enzymes is also provided in Table 1 and Table 2, herein.Additional cyanobacteria include members of the genus Chamaesiphon,Chroococcus, Cyanobacterium, Cyanobium, Cyanothece, Dactylococcopsis,Gloeobacter, Gloeocapsa, Gloeothece, Microcystis, Prochlorococcus,Prochloron, Synechococcus, Synechocystis, Cyanocystis, Dermocarpella,Stanieria, Xenococcus, Chroococcidiopsis, Myxosarcina, Arthrospira,Borzia, Crinalium, Geitlerinemia, Leptolyngbya, Limnothrix, Lyngbya,Microcoleus, Oscillatoria, Planktothrix, Prochlorothrix, Pseudanabaena,Spirulina, Starria, Symploca, Trichodesmium, Tychonema, Anabaena,Anabaenopsis, Aphanizomenon, Cyanospira, Cylindrospermopsis,Cylindrospermum, Nodularia, Nostoc, Scylonema, Calothrix, Rivularia,Tolypothrix, Chlorogloeopsis, Fischerella, Geitieria, Iyengariella,Nostochopsis, Stigonema and Thermosynechococcus.

Green non-sulfur bacteria include but are not limited to the followinggenera: Chloroflexus, Chloronema, Oscillochloris, Heliothrix,Herpetosiphon, Roseiflexus, and Thermomicrobium.

Green sulfur bacteria include but are not limited to the followinggenera:

Chlorobium, Clathrochloris, and Prosthecochloris.

Purple sulfur bacteria include but are not limited to the followinggenera: Allochromatium, Chromatium, Halochromatium, Isochromatium,Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa,Thiorhodococcus, and Thiocystis,

Purple non-sulfur bacteria include but are not limited to the followinggenera: Phaeospirillum, Rhodobaca, Rhodobacter, Rhodomicrobium,Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodospirillum,Rodovibrio, and Roseospira.

Aerobic chemolithotrophic bacteria include but are not limited tonitrifying bacteria such as Nitrobacteraceae sp., Nitrobacter sp.,Nitrospina sp., Nitrococcus sp., Nitrospira sp., Nitrosomonas sp.,Nitrosococcus sp., Nitrosospira sp., Nitrosolobus sp., Nitrosovibriosp.; colorless sulfur bacteria such as, Thiovulum sp., Thiobacillus sp.,Thiomicrospira sp., Thiosphaera sp., Thermothrix sp.; obligatelychemolithotrophic hydrogen bacteria such as Hydrogenobacter sp., ironand manganese-oxidizing and/or depositing bacteria such as Siderococcussp., and magnetotactic bacteria such as Aquaspirillum sp.

Archaeobacteria include but are not limited to methanogenicarchaeobacteria such as Methanobacterium sp., Methanobrevibacter sp.,Methanothermus sp., Methanococcus sp., Methanomicrobium sp.,Methanospirillum sp., Methanogenium sp., Methanosarcina sp.,Methanolobus sp., Methanothrix sp., Methanococcoides sp., Methanoplanussp.; extremely thermophilic S-Metabolizers such as Thermoproteus sp.,Pyrodictium sp., Sulfolobus sp., Acidianus sp. and other microorganismssuch as, Bacillus subtilis, Saccharomyces cerevisiae, Streptomyces sp.,Ralstonia sp., Rhodococcus sp., Corynebacteria sp., Brevibacteria sp.,Mycobacteria sp., and oleaginous yeast.

Preferred organisms for the manufacture of n-alkanes according to themethods disclosed herein include: Arabidopsis thaliana, Panicumvirgatum, Miscanthus giganteus, and Zea mays (plants); Botryococcusbraunii, Chlamydomonas reinhardtii and Dunaliela salina (algae);Synechococcus sp PCC 7002, Synechococcus sp. PCC 7942, Synechocystis sp.PCC 6803, Thermosynechococcus elongatus BP-1 (cyanobacteria); Chlorobiumtepidum (green sulfur bacteria), Chloroflexus auranticus (greennon-sulfur bacteria); Chromatium tepidum and Chromatium vinosum (purplesulfur bacteria); Rhodospirillum rubrum, Rhodobacter capsulatus, andRhodopseudomonas palusris (purple non-sulfur bacteria).

Yet other suitable organisms include synthetic cells or cells producedby synthetic genomes as described in Venter et al. US Pat. Pub. No.2007/0264688, and cell-like systems or synthetic cells as described inGlass et al. US Pat. Pub. No. 2007/0269862.

Still, other suitable organisms include microorganisms that can beengineered to fix carbon dioxide bacteria such as Escherichia coli,Acetobacter aceti, Bacillus subtilis, yeast and fungi such asClostridium ljungdahlii, Clostridium thermocellum, Penicilliumchrysogenum, Pichia pastoris, Saccharomyces cerevisiae,Schizosaccharomyces pombe, Pseudomonas fluorescens, or Zymomonasmobilis.

A suitable organism for selecting or engineering is autotrophic fixationof CO₂ to products. This would cover photosynthesis and methanogenesis.Acetogenesis, encompassing the three types of CO₂ fixation; Calvincycle, acetyl-CoA pathway and reductive TCA pathway is also covered. Thecapability to use carbon dioxide as the sole source of cell carbon(autotrophy) is found in almost all major groups of prokaryotes. The CO₂fixation pathways differ between groups, and there is no cleardistribution pattern of the four presently-known autotrophic pathways.See, e.g., Fuchs, G. 1989. Alternative pathways of autotrophic CO ₂fixation, p. 365-382. In H. G. Schlegel, and B. Bowien (ed.),Autotrophic bacteria. Springer-Verlag, Berlin, Germany. The reductivepentose phosphate cycle (Calvin-Bassham-Benson cycle) represents the CO₂fixation pathway in almost all aerobic autotrophic bacteria, forexample, the cyanobacteria.

For producing n-alkanes via the recombinant expression of AAR and/or ADMenzymes, an engineered cyanobacteria, e.g., a Synechococcus orThermosynechococcus species, is preferred. Other preferred organismsinclude Synechocystis, Klebsiella oxytoca, Escherichia coli orSaccharomyces cerevisiae. Other prokaryotic, archaea and eukaryotic hostcells are also encompassed within the scope of the present invention.

Carbon-Based Products of Interest: Hydrocarbons & Alcohols

In various embodiments of the invention, desired hydrocarbons and/oralcohols of certain chain length or a mixture thereof can be produced.In certain aspects, the host cell produces at least one of the followingcarbon-based products of interest: 1-dodecanol, 1-tetradecanol,1-pentadecanol, n-tridecane, n-tetradecane, 15:1 n-pentadecane,n-pentadecane, 16:1 n-hexadecene, n-hexadecane, 17:1 n-heptadecene,n-heptadecane, 16:1 n-hexadecen-ol, n-hexadecan-1-ol andn-octadecen-1-ol, as shown in the Examples herein. In other aspects, thecarbon chain length ranges from C₁₀ to C₂₀. Accordingly, the inventionprovides production of various chain lengths of alkanes, alkenes andalkanols suitable for use as fuels & chemicals.

In preferred aspects, the methods provide culturing host cells fordirect product secretion for easy recovery without the need to extractbiomass. These carbon-based products of interest are secreted directlyinto the medium. Since the invention enables production of variousdefined chain length of hydrocarbons and alcohols, the secreted productsare easily recovered or separated. The products of the invention,therefore, can be used directly or used with minimal processing.

Fuel Compositions

In various embodiments, compositions produced by the methods of theinvention are used as fuels. Such fuels comply with ASTM standards, forinstance, standard specifications for diesel fuel oils D 975-09b, andJet A, Jet A-1 and Jet B as specified in ASTM Specification D. 1655-68.Fuel compositions may require blending of several products to produce auniform product. The blending process is relatively straightforward, butthe determination of the amount of each component to include in a blendis much more difficult. Fuel compositions may, therefore, includearomatic and/or branched hydrocarbons, for instance, 75% saturated and25% aromatic, wherein some of the saturated hydrocarbons are branchedand some are cyclic. Preferably, the methods of the invention produce anarray of hydrocarbons, such as C₁₃-C₁₇ or C₁₀-C₁₅ to alter cloud point.Furthermore, the compositions may comprise fuel additives, which areused to enhance the performance of a fuel or engine. For example, fueladditives can be used to alter the freezing/gelling point, cloud point,lubricity, viscosity, oxidative stability, ignition quality, octanelevel, and flash point. Fuels compositions may also comprise, amongothers, antioxidants, static dissipater, corrosion inhibitor, icinginhibitor, biocide, metal deactivator and thermal stability improver.

In addition to many environmental advantages of the invention such asCO₂ conversion and renewable source, other advantages of the fuelcompositions disclosed herein include low sulfur content, low emissions,being free or substantially free of alcohol and having high cetanenumber.

Carbon Fingerprinting

Biologically-produced carbon-based products, e.g., ethanol, fatty acids,alkanes, isoprenoids, represent a new commodity for fuels, such asalcohols, diesel and gasoline. Such biofuels have not been producedusing biomass but use CO2 as its carbon source. These new fuels may bedistinguishable from fuels derived form petrochemical carbon on thebasis of dual carbon-isotopic fingerprinting. Such products,derivatives, and mixtures thereof may be completely distinguished fromtheir petrochemical derived counterparts on the basis of ¹⁴C (fM) anddual carbon-isotopic fingerprinting, indicating new compositions ofmatter.

There are three naturally occurring isotopes of carbon: ¹²C, ¹³C, and¹⁴C. These isotopes occur in above-ground total carbon at fractions of0.989, 0.011, and 10⁻¹², respectively. The isotopes ¹²C and ¹³C arestable, while ¹⁴C decays naturally to ¹⁴N, a beta particle, and ananti-neutrino in a process with a half-life of 5730 years. The isotope¹⁴C originates in the atmosphere, due primarily to neutron bombardmentof ¹⁴N caused ultimately by cosmic radiation. Because of its relativelyshort half-life (in geologic terms), ¹⁴C occurs at extremely low levelsin fossil carbon. Over the course of 1 million years without exposure tothe atmosphere, just 1 part in 10⁵⁰ will remain ¹⁴C.

The ¹³C:¹²C ratio varies slightly but measurably among natural carbonsources. Generally these differences are expressed as deviations fromthe ¹³C:¹²C ratio in a standard material. The international standard forcarbon is Pee Dee Belemnite, a form of limestone found in SouthCarolina, with a ¹³C fraction of 0.0112372. For a carbon source a, thedeviation of the ¹³C:¹²C ratio from that of Pee Dee Belemnite isexpressed as: δ_(a)=(R_(a)/R_(s))−1, where R_(a)=¹³C:¹²C ratio in thenatural source, and R_(s)=¹³C:¹²C ratio in Pee Dee Belemnite, thestandard. For convenience, δ_(a) is expressed in parts per thousand, or%

. A negative value of δ_(a) shows a bias toward ¹²C over ¹³C as comparedto Pee Dee Belemnite. Table A shows δ_(a) and ¹⁴C fraction for severalnatural sources of carbon.

TABLE A 13C:12C variations in natural carbon sources Source −δ_(a) (‰)References Underground coal 32.5 Farquhar et al. (1989) Plant Mol.Biol., 40:503-37 Fossil fuels 26 Farquhar et al. (1989) Plant Mol.Biol., 40:503-37 Ocean DIC* 0-1.5 Goericke et al. (1994) Chapter 9 inStable Isotopes in Ecology and Environmental Science, by K. Lajtha andR. H. Michener, Blackwell Publishing; Ivlev (2010) Separation Sci.Technol. 36:1819-1914 Atmospheric 6-8   Ivlev (2010) Separation Sci. CO2Technol. 36:1819-1914; Farquhar et al. (1989) Plant Mol. Biol.,40:503-37 Freshwater DIC* 6-14  Dettman et al. (1999) Geochim.Cosmochim. Acta 63:1049-1057 Pee Dee 0 Ivlev (2010) Separation Sci.Belemnite Technol. 36:1819-1914 *DIC = dissolved inorganic carbon

Biological processes often discriminate among carbon isotopes. Thenatural abundance of ¹⁴C is very small, and hence discrimination for oragainst ¹⁴C is difficult to measure. Biological discrimination between¹³C and ¹²C, however, is well-documented. For a biological product p, wecan define similar quantities to those above: δ_(p)=(R_(p)/R_(s))−1,where R_(p)=¹³C:¹²C ratio in the biological product, and R_(s)=¹³C:¹²Cratio in Pee Dee Belemnite, the standard. Table B shows measureddeviations in the ¹³C:¹²C ratio for some biological products.

TABLE B ¹³C:¹²C variations in se1ected biological products Product−δ_(p) (‰) −D (‰)* References Plant sugar/starch 18-28 10-20 Ivlev(2010) Separation from atmospheric Sci. Technol. 36:1819- CO2 1914Cyanobacterial 18-31 16.5-31   Goericke et al. (1994) biomass fromChapter 9 in Stab1e marine DIC Isotopes in Ecology and EnvironmentalScience, by K. Lajtha and R. H. Michener, Blackwell Publishing; Sakataet al. (1997) Geochim. Cosmochim. Acta, 61:5379-89 Cyanobacterial 39-4037.5-40   Sakata et al. (1997) lipid from Geochim. Cosmochim. marine DICActa, 61:5379-89 Algal lipid from 17-28 15.5-28   Goericke et al. (1994)marine DIC Chapter 9 in Stable Isotopes in Ecology and EnvironmentalScience, by K. Lajtha and R. H. Michener, Blackwell Publishing; Abelseonet al. (1961) Proc. Natl. Acad. Sci., 47:623-32 Algal biomass from 17-36 3-30 Marty et al. (2008) freshwater DIC Limnol. Oceanogr.: Methods6:51-63 E. coli lipid from 15-27 near 0 Monson et al. (1980) J. plantsugar Biol. Chem., 255:11435-41 Cyanobacterial lipid 63.5-66   37.5-40  — from fossil carbon Cyanobacterial 42.5-57   16.5-31   — biomass fromfossil carbon *D = discrimination by a biological process in itsutilization of ¹²C vs. ¹³C (see text)

Table B introduces a new quantity, D. This is the discrimination by abiological process in its utilization of ¹²C vs. ¹³C. We define D asfollows: D=(R_(p)/R_(a))−1. This quantity is very similar to δ_(a) andδ_(p), except we now compare the biological product directly to thecarbon source rather than to a standard. Using D, we can combine thebias effects of a carbon source and a biological process to obtain thebias of the biological product as compared to the standard. Solving forδ_(p), we obtain: δ_(p)=(D)(δ_(a))+D+δ_(a), and, because (D)(δ_(a)) isgenerally very small compared to the other terms, δ_(p)≈δ_(a)+D.

For a biological product having a production process with a known D, wemay therefore estimate δ_(p) by summing δ_(a) and D. We assume that Doperates irrespective of the carbon source. This has been done in TableB for cyanobacterial lipid and biomass produced from fossil carbon. Asshown in the Table A and Table B, above, cyanobacterial products madefrom fossil carbon (in the form of, for example, flue gas or otheremissions) will have a higher δ_(p) than those of comparable biologicalproducts made from other sources, distinguishing them on the basis ofcomposition of matter from these other biological products. In addition,any product derived solely from fossil carbon will have a negligiblefraction of ¹⁴C, while products made from above-ground carbon will havea ¹⁴C fraction of approximately 10⁻¹².

Accordingly, in certain aspects, the invention provides variouscarbon-based products of interest characterized as −δ_(p)(%

) of about 63.5 to about 66 and −D(%

) of about 37.5 to about 40.

Antibodies

In another aspect, the present invention provides isolated antibodies,including fragments and derivatives thereof that bind specifically tothe isolated polypeptides and polypeptide fragments of the presentinvention or to one or more of the polypeptides encoded by the isolatednucleic acids of the present invention. The antibodies of the presentinvention may be specific for linear epitopes, discontinuous epitopes orconformational epitopes of such polypeptides or polypeptide fragments,either as present on the polypeptide in its native conformation or, insome cases, as present on the polypeptides as denatured, as, e.g., bysolubilization in SDS. Among the useful antibody fragments provided bythe instant invention are Fab, Fab′, Fv, F(ab′)₂, and single chain Fvfragments.

By “bind specifically” and “specific binding” is here intended theability of the antibody to bind to a first molecular species inpreference to binding to other molecular species with which the antibodyand first molecular species are admixed. An antibody is saidspecifically to “recognize” a first molecular species when it can bindspecifically to that first molecular species.

As is well known in the art, the degree to which an antibody candiscriminate as among molecular species in a mixture will depend, inpart, upon the conformational relatedness of the species in the mixture;typically, the antibodies of the present invention will discriminateover adventitious binding to unrelated polypeptides by at leasttwo-fold, more typically by at least 5-fold, typically by more than10-fold, 25-fold, 50-fold, 75-fold, and often by more than 100-fold, andon occasion by more than 500-fold or 1000-fold.

Typically, the affinity or avidity of an antibody (or antibody multimer,as in the case of an IgM pentamer) of the present invention for apolypeptide or polypeptide fragment of the present invention will be atleast about 1×10⁻⁶ M, typically at least about 5×10⁻⁷ M, usefully atleast about 1×10⁻⁷ M, with affinities and avidities of 1×10⁻8 M, 5×10⁻⁹M, 1×10⁻¹⁰ M and even stronger proving especially useful.

The isolated antibodies of the present invention may benaturally-occurring forms, such as IgG, IgM, IgD, IgE, and IgA, from anymammalian species. For example, antibodies are usefully obtained fromspecies including rodents-typically mouse, but also rat, guinea pig, andhamster-lagomorphs, typically rabbits, and also larger mammals, such assheep, goats, cows, and horses. The animal is typically affirmativelyimmunized, according to standard immunization protocols, with thepolypeptide or polypeptide fragment of the present invention.

Virtually all fragments of 8 or more contiguous amino acids of thepolypeptides of the present invention may be used effectively asimmunogens when conjugated to a carrier, typically a protein such asbovine thyroglobulin, keyhole limpet hemocyanin, or bovine serumalbumin, conveniently using a bifunctional linker. Immunogenicity mayalso be conferred by fusion of the polypeptide and polypeptide fragmentsof the present invention to other moieties. For example, peptides of thepresent invention can be produced by solid phase synthesis on a branchedpolylysine core matrix; these multiple antigenic peptides (MAPs) providehigh purity, increased avidity, accurate chemical definition andimproved safety in vaccine development. See, e.g., Tam et al., Proc.Natl. Acad. Sci. USA 85:5409-5413 (1988); Posnett et al., J. Biol. Chem.263, 1719-1725 (1988).

Protocols for immunization are well-established in the art. Suchprotocols often include multiple immunizations, either with or withoutadjuvants such as Freund's complete adjuvant and Freund's incompleteadjuvant. Antibodies of the present invention may be polyclonal ormonoclonal, with polyclonal antibodies having certain advantages inimmuno-histochemical detection of the proteins of the present inventionand monoclonal antibodies having advantages in identifying anddistinguishing particular epitopes of the proteins of the presentinvention. Following immunization, the antibodies of the presentinvention may be produced using any art-accepted technique. Host cellsfor recombinant antibody production—either whole antibodies, antibodyfragments, or antibody derivatives—can be prokaryotic or eukaryotic.Prokaryotic hosts are particularly useful for producing phage displayedantibodies, as is well known in the art. Eukaryotic cells, includingmammalian, insect, plant and fungal cells are also useful for expressionof the antibodies, antibody fragments, and antibody derivatives of thepresent invention. Antibodies of the present invention can also beprepared by cell free translation.

The isolated antibodies of the present invention, including fragmentsand derivatives thereof, can usefully be labeled. It is, therefore,another aspect of the present invention to provide labeled antibodiesthat bind specifically to one or more of the polypeptides andpolypeptide fragments of the present invention. The choice of labeldepends, in part, upon the desired use. In some cases, the antibodies ofthe present invention may usefully be labeled with an enzyme.Alternatively, the antibodies may be labeled with colloidal gold or witha fluorophore. For secondary detection using labeled avidin,streptavidin, captavidin or neutravidin, the antibodies of the presentinvention may usefully be labeled with biotin. When the antibodies ofthe present invention are used, e.g., for Western blotting applications,they may usefully be labeled with radioisotopes, such as ³³P, ³²P, ³⁵S,³H and ¹²⁵I. As would be understood, use of the labels described aboveis not restricted to any particular application.

The following examples are for illustrative purposes and are notintended to limit the scope of the present invention.

Example 1

A pathway for the enzymatic synthesis of n-alkanes. An enzymatic processfor the production of n-alkanes in, e.g., cyanobacteria is shown in FIG.1A based on the sequential activity of (1) an AAR enzyme, e.g., tll1312,an acyl-ACP reductase; and (2) an ADM enzyme, e.g., tll1313, a putativealkanal decarboxylative monooxygenase, that uses reduced ferredoxin aselectron donor. The AAR activity is distinct from the relatively wellcharacterized acyl-CoA reductase activity exhibited by proteins such asAcr1 from Acinetobacter calcoaceticus (Reiser S and Somerville C (1997)J. Bacteriol. 179:2969-2975). A membranous ADM activity has previouslybeen identified in insect microsomal preparations (Reed J R et al.(1994) Proc. Natl. Acad. Sci. USA 91:10000-10004; Reed J R et al. (1995)Musca domestica. Biochemistry 34:16221-16227).

FIGS. 1B and 1C summarize the names and activities of the enzymesinvolved in the biosynthesis of n-alkanals. FIG. 1B depicts therelatively well characterized acyl-CoA reductase activity (EC 1.2.1.50)exhibited by proteins such as Acr1 from Acinetobacter calcoaceticus. InFIG. 1C, the two well-known ACP-related reductases that are involved infatty acid biosynthesis, β-ketoacyl-ACP reductase (EC 1.1.1.100) andenoyl-ACP reductase (EC 1.3.1.9, 1.3.1.10), are contrasted with theacyl-ACP reductase (AAR) (no EC number yet assigned) believed to beinvolved in the biosynthetic pathway for n-alkanes in cyanobacteria. Thekey difference between AAR and acyl-CoA reductase (EC 1.2.1.50) is thatACP is the acyl carrier rather than coenzyme A. Supporting thisdistinction, it has been shown that acyl-CoA reductase Acr1 fromAcinetobacter calcoaceticus can only generate alkanals from acyl-CoA andnot acyl-ACP (Resier S and Somerville C (1997) J Bacteriol. 179:2969-2975).

ADM also lacks a presently assigned EC number. An alkanal monooxygenase(EC 1.14.14.3), often referred to as luciferase, is known to catalyzethe conversion of n-alkanal to n-alkanoic acid. This activity isdistinct from the ADM activity (n-alkanal to (n−1)-alkane) proposedherein, although both use n-alkanal and molecular oxygen as substrates.

Cyanobacterial AAR and ADM homologs for production of n-alkanes. In thisexample, homologs of cyanobacterial AAR and ADM genes (e.g., homologs ofSynechococcus elongatus PCC 7942 SYNPCC7942_(—)1594 and/orSYNPCC7942_(—)1593 protein, respectively) are identified using a BLASTsearch. These proteins can be expressed in a variety of organisms(bacteria, yeast, plant, etc.) for the purpose of generating andisolating n-alkanes and other desired carbon-based products of interestfrom the organisms. A search of the non-redundant BLAST protein databaserevealed counterparts for each protein in other cyanobacteria.

To determine the degree of similarity among homologs of theSynechococcus elongatus PCC 7942 SYNPCC7942_(—)1594 protein, the341-amino acid protein sequence was queried using BLAST(http://blast.ncbi.nlm.nih.gov/) against the “nr” non-redundant proteindatabase. Homologs were taken as matching proteins whose alignments (i)covered >90% length of SYNPCC7942_(—)1594, (ii) covered >90% of thelength of the matching protein, and (iii) had >50% identity withSYNPCC7942_(—)1594 (Table 1).

TABLE 1 Protein homologs of SYNPCC7942_1594 (AAR) SEQ BLAST ID Score,Organism NO: Homolog accession # E-value Synechococcus elongatus 6(SYNPCC7942_1594) n/a PCC 7942 Synechococcus elongatus 23 YP_400611.1706, 0.0 PCC 7942 [cyanobacteria] taxid 1140 Synechococcus elongatus 24YP_170761.1 706, 0.0 PCC 6301 [cyanobacteria] taxid 269084 Anabaenavariabilis 25 YP_323044.1 538, 4e-151 ATCC 29413 [cyanobacteria] taxid240292 Nostoc sp. PCC 7120 26 NP_489324.1 535, 3e-150 [cyanobacteria]taxid 103690 ‘Nostoc azollae’ 0708 27 ZP_03763674.1 533, 1e-149[cyanobacteria] taxid 551115 Cyanothece sp. PCC 7425 28 YP_002481152.1526, 9e-148 [cyanobacteria] taxid 395961 Nodularia spumigena 29ZP_01628095.1 521, 3e-146 CCY 9414 [cyanobacteria] taxid 313624 Lyngbyasp. PCC 8106 30 ZP_01619574.1 520, 6e-146 [cyanobacteria] taxid 313612Nostoc punctiforme PCC 31 YP_001865324.1 520, 7e-146 73102[cyanobacteria] taxid 63737 Trichodesmium 32 YP_721978.1 517, 6e-145erythraeum IMS101 [cyanobacteria] taxid 203124 Thermosynechococcus 2NP_682102.1 516, 2e-144 elongatus BP-1 [cyanobacteria] taxid 197221Acaryochloris marina 33 YP_001518341.1 512, 2e-143 MBIC11017[cyanobacteria] taxid 329726 Cyanothece sp. PCC 8802 34 ZP_03142196.1510, 8e-143 [cyanobacteria] taxid 395962 Cyanothece sp. PCC 8801 35YP_002371106.1 510, 8e-143 [cyanobacteria] taxid 41431 Microcoleus 36YP_002619867.1 509, 2e-142 chthonoplastes PCC 7420 [cyanobacteria] taxid118168 Arthrospira maxima 37 ZP_03273554.1 507, 7e-142 CS-328[cyanobacteria] taxid 513049 Synechocystis sp. 38 NP_442146.1 504,5e-141 PCC 6803 [cyanobacteria] taxid 1148 Cyanothece sp. CCY 0110 39ZP_01728620.1 501, 4e-140 [cyanobacteria] taxid 391612 Synechococcus sp.40 YP_002711557.1 500, 1e-139 PCC 7335 [cyanobacteria] taxid 91464Cyanothece sp. 41 YP_001802846.1 489, 2e-136 ATCC 51142 [cyanobacteria]taxid 43989 Gloeobacter violaceus 42 NP_926091.1 487, 7e-136 PCC 7421[cyanobacteria] taxid 251221 Microcystis aeruginosa 43 YP_001660322.1486, 1e-135 NIES-843 [cyanobacteria] taxid 449447 Crocosphaera watsonii44 ZP_00516920.1 486, 1e-135 WH 8501 [cyanobacteria] taxid 165597Microcystis aeruginosa 45 emb|CA090781.1 484, 8e-135 PCC 7806[cyanobacteria] taxid 267872 Synechococcus sp. 46 ZP_01085337.1 471,4e-131 WH 5701 [cyanobacteria] taxid 69042 Synechococcus sp. 47YP_001227841.1 464, 8e-129 RCC307 [cyanobacteria] taxid 316278uncultured marine type-A 48 gb|ABD96327.1 462 , 2e-128 Synechococcus GOM306 [cyanobacteria] taxid 364150 Synechococcus sp. 49 NP_897828.1 462,2e-128 WH 8102 [cyanobacteria] taxid 84588 Synechococcus sp. 50YP_001224378.1 459, 2e-127 WH 7803 [cyanobacteria] taxid 32051uncultured marine type-A 51 gb|ABD96480.1 458, 3e-127 Synechococcus GOM5D20[cyanobacteria] taxid 364154 Synechococcus sp. 52 ZP_01123215.1 457,5e-127 WH 7805 [cyanobacteria] taxid 59931 uncultured marine type-A 53gb|ABB92249.1 457, 8e-127 Synechococcus 5B2 [cyanobacteria] taxid 359140Synechococcus sp. 54 ZP_01079773.1 456, 2e-126 RS9917 [cyanobacteria]taxid 221360 Synechococcus sp. 55 YP_377636.1 454, 6e-126 CC9902[cyanobacteria] taxid 316279 Prochlorococcus marinus 56 NP_874926.1 453,9e-126 subsp. marinus str. CCMP1375 [cyanobacteria] taxid 167539Prochlorococcus 57 NP_895058.1 453, 1e-125 marinus str. MIT 9313[cyanobacteria] taxid 74547 uncultured marine type-A 58 gb|ABD96274.1452, 2e-125 Synechococcus GOM 3M9 [cyanobacteria] taxid 364149uncultured marine type-A 59 gb|ABD96442.1 452, 2e-125 Synechococcus GOM4P21 [cyanobacteria] taxid 364153 Synechococcus sp. BL107 60ZP_01469469.1 452, 2e-125 [cyanobacteria] taxid 313625 Cyanobium sp. PCC7001 61 YP_002597253.1 451, 4e-125 [cyanobacteria] taxid 180281Prochlorococcus 62 YP_001014416.1 449, 2e-124 marinus str. NATL1A[cyanobacteria] taxid 167555 Prochlorococcus 63 YP_001010913.1 447,6e-124 marinus str. MIT 9515 [cyanobacteria] taxid 167542 Synechococcussp. 64 YP_381056.1 447, 8e-124 CC9605 [cyanobacteria] taxid 110662Prochlorococcus 65 YP_001550421.1 446, 2e-123 marinus str. MIT 9211[cyanobacteria] taxid 93059 Prochlorococcus marinus 66 NP_892651.1 446,2e-123 subsp. pastoris str. CCMP1986 [cyanobacteria] taxid 59919Prochlorococcus 67 YP_001090783.1 445, 3e-123 marinus str. MIT 9301[cyanobacteria] taxid 167546 Synechococcus sp. 68 ZP_01472595.1 445,3e-123 RS9916 [cyanobacteria] taxid 221359 Prochlorococcus 69YP_293055.1 445, 4e-123 marinus str. NATL2A [cyanobacteria] taxid 59920Prochlorococcus 70 YP_002673377.1 444, 7e-123 marinus str. MIT 9202[cyanobacteria] taxid 93058 Synechococcus sp. 71 YP_731192.1 443, 1e-122CC9311 [cyanobacteria] taxid 64471 Prochlorococcus 72 YP_001483815.1442, 2e-122 marinus str. MIT 9215 [cyanobacteria] taxid 93060Prochlorococcus 73 YP_001008982.1 442, 3e-122 marinus str. AS9601[cyanobacteria] taxid 146891 Synechococcus sp. 74 YP_473896.1 441,5e-122 JA-3-3Ab [cyanobacteria] taxid 321327 Synechococcus sp. JA-2- 75YP_478638.1 440, 8e-122 3B′a(2-13) [cyanobacteria] taxid 321332Prochlorococcus 76 YP_397030.1 436, 1e-120 marinus str. MIT 9312[cyanobacteria] taxid 74546

To determine the degree of similarity among homologs of theSynechococcus elongatus PCC 7942 SYNPCC7942_(—)1593 protein, the 231amino acid protein sequence was queried using BLAST(http://blast.ncbi.nlm.nih.gov/) against the “nr” non-redundant proteindatabase. Homologs were taken as matching proteins whose alignments (i)covered >90% length of SYNPCC7942_(—)1593, (ii) covered >90% of thelength of the matching protein, (iii) and had >50% identity withSYNPCC7942_(—)1593 (Table 2).

TABLE 2 Protein homologs of SYNPCC7942_1593 (ADM) SEQ BLAST ID Score,Organism NO: Homolog accession # E-value Synechococcus 8 (SYNPCC7942_1593 ) n/a elongatus PCC 7942 [cyanobacteria] Synechococcus 77YP_400610.1 475, 1e-132 elongatus PCC 7942 [cyanobacteria] taxid 1140Synechococcus elongatus 78 YP_170760.1 475, 2e-132 PCC 6301[cyanobacteria] taxid 269084 Arthrospira maxima 79 ZP_03273549.1 378,3e-103 CS-328 [cyanobacteria] taxid 513049 Microcoleus 80 YP_002619869.1376, 1e-102 chthonoplastes PCC 7420 [cyanobacteria] taxid 118168 Lyngbyasp. PCC 8106 81 ZP_01619575.1 374, 5e-102 [cyanobacteria] taxid 313612Nodularia spumigena 82 ZP_01628096.1 369, 1e-100 CCY 9414[cyanobacteria] taxid 313624 Microcystis aeruginosa 83 YP_001660323.1367, 5e-100 NIES-843 [cyanobacteria] taxid 449447 Microcystis aeruginosa84 emb|CA090780.1 364, 3e-99 PCC 7806 [cyanobacteria] taxid 267872Nostoc sp. PCC 7120 85 NP_489323.1 363, 1e-98 [cyanobacteria] taxid103690 Anabaena variabilis 86 YP_323043.1 362, 2e-98 ATCC 29413[cyanobacteria] taxid 240292 Crocosphaera watsonii 87 ZP_00514700.1 359,1e-97 WH 8501 [cyanobacteria] taxid 165597 Trichodesmium erythraeum 88YP_721979.1 358, 2e-97 IMS101 [cyanobacteria] taxid 203124 Synechococcussp. 89 YP_002711558.1 357, 6e-97 PCC 7335 [cyanobacteria] taxid 91464‘Nostoc azollae’ 0708 90 ZP_03763673.1 355, 3e-96 [cyanobacteria] taxid551115 Synechocystis sp. 91 NP_442147.1 353, 5e-96 PCC 6803[cyanobacteria] taxid 1148 Cyanothece sp. 92 YP_001802195.1 352, 2e-95ATCC 51142 [cyanobacteria] taxid 43989 Cyanothece sp. 93 ZP_01728578.1352, 2e-95 CCY 0110 [cyanobacteria] taxid 391612 Cyanothece sp. 94YP_002481151.1 350, 7e-95 PCC 7425 [cyanobacteria] taxid 395961 Nostocpunctiforme 95 YP_001865325.1 349, 1e-94 PCC 73102 [cyanobacteria] taxid63737 Acaryochloris marina 96 YP_001518340.1 344, 4e-93 MBIC11017[cyanobacteria] taxid 329726 Cyanothece sp. 97 ZP_03142957.1 342, 1e-92PCC 8802 [cyanobacteria] taxid 395962 Cyanothece sp. 98 YP_002370707.1342, 1e-92 PCC 8801 [cyanobacteria] taxid 41431 Thermosynechococcus 4NP_682103.1 332, 2e-89 elongatus BP-1 [cyanobacteria] taxid 197221Synechococcus sp. 99 YP_478639.1 319, 1e-85 JA-2-3B′a(2-13)[cyanobacteria] taxid 321332 Synechococcus sp. 100 YP_001227842.1 319,1e-85 RCC307 [cyanobacteria] taxid 316278 Synechococcus sp. 101YP_001224377.1 313, 8e-84 WH 7803 [cyanobacteria] taxid 32051Synechococcus sp. 102 NP_897829.1 311, 3e-83 WH 8102 [cyanobacteria]taxid 84588 Synechococcus sp. 103 ZP_01123214.1 310, 6e-83 WH 7805[cyanobacteria] taxid 59931 uncultured marine type-A 104 gb|ABD96376.1309, 1e-82 Synechococcus GOM 3012 [cyanobacteria] taxid 364151Synechococcus sp. 105 YP_473897.1 309, 1e-82 JA-3-3Ab [cyanobacteria]taxid 321327 uncultured marine type-A 106 gb|ABD96328.1 309, 1e-82Synechococcus GOM 306 [cyanobacteria] taxid 364150 uncultured marinetype-A 107 gb|ABD96275.1 308, 2e-82 Synechococcus GOM 3M9[cyanobacteria] taxid 364149 Synechococcus sp. 108 YP_731193.1 306,7e-82 CC9311 [cyanobacteria] taxid 64471 uncultured marine type-A 109gb|ABB92250.1 306, 9e-82 Synechococcus 5B2 [cyanobacteria] taxid 359140Synechococcus sp. 110 ZP_01085338.1 305, 3e-81 WH 5701 [cyanobacteria]taxid 69042 Gloeobacter violaceus 111 NP_926092.1 303, 8e-81 PCC 7421[cyanobacteria] taxid 251221 Synechococcus sp. 112 ZP_01472594.1 303,9e-81 RS9916 [cyanobacteria] taxid 221359 Synechococcus sp. 113ZP_01079772.1 300, 6e-80 RS9917 [cyanobacteria] taxid 221360Synechococcus sp. 114 YP_381055.1 300, 7e-80 CC9605 [cyanobacteria]taxid 110662 Prochlorococcus 115 YP_001016795.1 294, 4e-78 marinus str.MIT 9303 [cyanobacteria] taxid 59922 Cyanobium sp. 116 YP_002597252.1294, 6e-78 PCC 7001 [cyanobacteria] taxid 180281 Prochlorococcus 117NP_895059.1 291, 3e-77 marinus str. MIT 9313 [cyanobacteria] taxid 74547Synechococcus sp. 118 YP_377637.1 289, 1e-76 CC9902 [cyanobacteria]taxid 316279 Prochlorococcus 119 YP_001090782.1 287, 5e-76 marinus str.MIT 9301 [cyanobacteria] taxid 167546 Synechococcus sp. 120ZP_01469468.1 287, 6e-76 BL107 [cyanobacteria] taxid 313625Prochlorococcus 121 YP_001008981.1 286, 2e-75 marinus str. AS9601[cyanobacteria] taxid 146891 Prochlorococcus 12 YP_397029.1 282, 1e-74marinus str. MIT 9312 [cyanobacteria] taxid 74546 Prochlorococcus 122NP_892650.1 280, 9e-74 marinus subsp. pastoris str. CCMP1986[cyanobacteria] taxid 59919 Prochlorococcus 123 YP_001550420.1 279,2e-73 marinus str. MIT 9211 [cyanobacteria] taxid 93059 Prochlorococcus124 YP_293054.1 276, 9e-73 marinus str. NATL2A [cyanobacteria] taxid59920 Prochlorococcus 125 YP_001014415.1 276, 9e-73 marinus str. NATL1A[cyanobacteria] taxid 167555 Prochlorococcus 126 NP_874925.1 276, 1e-72marinus subsp. marinus str. CCMP1375 [cyanobacteria] taxid 167539Prochlorococcus 127 YP_001010912.1 273, 6e-72 marinus str. MIT 9515[cyanobacteria] taxid 167542 Prochlorococcus 128 YP_01483814.1 273,9e-72 marinus str. MIT 9215 [cyanobacteria] taxid 93060

The amino acid sequences referred to in the Table, as those sequencesappeared in the NCBI database on Jul. 9, 2009, by accession number areincorporated by reference herein.

An AAR enzyme from Table 1, and/or an ADM enzyme from Table 2, or bothcan be expressed in a host cell of interest, wherein the host may be aheterologous host or the native host, i.e., the species from which thegenes were originally derived. In one embodiment, the invention providesa method of imparting n-alkane synthesis capability in a heterologousorganism, lacking native homologs of AAR and/or ADM, by engineering theorganism to express a gene encoding one of the enzymes listed in Table 1or Table 2. Also provided are methods of modulating n-alkane synthesisin an organism which already expresses one or both of the AAR and ADMenzymes by increasing the expression of the native enzymes, or byaugmenting native gene expression by the recombinant expression ofheterologous AAR and/or ADM enzymes. In addition, the invention providesmethods of modulating the degree of alkane synthesis by varying certainparameters, including the identity and/or compatibility of electrondonors, culture conditions, promoters for expressing AAR and/or ADMenzymes, and the like.

If the host lacks a suitable electron donor or lacks sufficient levelsof a suitable electron donor to achieve production of the desired amountof n-alkane, such electron donor may also be introduced recombinantly.Guidelines for optimizing electron donors for the reaction catalyzed bythe recombinant ADM proteins described herein may be summarized asfollows:

-   -   1. In cyanobacteria, electrons are shuttled from photosystem Ito        ferredoxin and from ferredoxin to the ADM enzyme.    -   2. In bacteria that lack photosystem I, electrons can be        shuttled from NADPH to ferredoxin via the action of        ferredoxin-NADP+reductase (EC 1.18.1.2) and from ferredoxin to        the ADM enzyme.    -   3. In bacteria that lack photosystem I, electrons can be        shuttled from NADPH to flavodoxin via the action of        ferredoxin-NADP+reductase (EC 1.18.1.2) and from flavodoxin to        the ADM enzyme.    -   4. In bacteria that lack photosystem I, electrons can be        shuttled from NADH to ferredoxin via the action of Trichomonas        vaginalis NADH dehydrogenase and from ferredoxin to the ADM        enzyme.    -   5. In all bacteria, electrons can be shuttled from pyruvate to        ferredoxin by the action of pyruvate:ferredoxin oxidoreductase        (EC 1.2.7.1), and from ferredoxin to the ADM enzyme.

In addition to the in vivo production of n-alkanes discussed above, AARand ADM proteins encoded by the genes listed in Tables 1 and 2 can bepurified. When incubated in vitro with an appropriate electron donor(e.g., a ferredoxin, as discussed above), the proteins will catalyze theenzymatic synthesis of n-alkanes in vitro from appropriate startingmaterials (e.g., an acyl-ACP or n-alkanal).

In addition to the pathways for n-alkane synthesis described above, theinvention also provides an alternative pathway, namely,acyl-CoA→n-alkanal (n−1)-alkane, via the successive activities ofacyl-CoA reductase (ACR) and ADM. Normally, acyl-CoA is the firstintermediate in metabolic pathways of fatty acid oxidation; thus, uponimport into the cell, exogenously added free fatty acids are convertedto acyl-CoAs by acyl-CoA synthetase (FIG. 1B). Acyl-CoA can also bederived purely biosynthetically as follows: acyl-ACP→free fattyacid→acyl-CoA, via the activities of cytoplasmic acyl-ACP thioesterase(EC 3.1.2.14; an example is leader-signal-less E. coli TesA) and theendogenous and/or heterologous acyl-CoA synthetase. Thus, in oneembodiment, the invention provides a method for the biosynthesis ofn-alkanes via the pathway: acyl-ACP→intracellular free fattyacid→acyl-CoA→n-alkanal→(n−1)-alkane (FIG. 1D), catalyzed by thesuccessive activities of acyl-ACP thioesterase, acyl-CoA synthetase,acyl-CoA reductase, and ADM. For example, the acyl-CoA reductase Acr1from Acinetobacter calcoaceticus and the ADM from Synechococcus sp.PCC7942 (SYNPCC7942_(—)1593) can be used to transform E. coli, which iscultured in the presence of exogenous free fatty acids. The free fattyacids are taken up by the cells as acyl-CoA, which are then converted ton-alkanal by Acr1, and thence to (n−1)-alkane by ADM.

Example 2 Production of n-Alkanes, n-Alkenes, and Fatty Alcohols inEscherichia coli K-12 through Heterologous Expression of Synechococcuselongatus PCC7942 SYNPCC7942_(—)1593 (adm) and SYNPCC7942_(—)1594 (aar)

The natural SYNPCC7942_(—)1593-SYNPCC7942_(—)1594 operonic sequence wasPCR-amplified from the genomic DNA of Synechococcus elongatus PCC7942and cloned into the pAQ1 homologous recombination vector pJB5 via NdeIand EcoRI. The resulting plasmid was denoted pJB823. This constructplaced the SYNPCC7942_(—)1593-SYNPCC7942_(—)1594 operon under thetranscriptional control of the constitutive aphII promoter. The sequenceof pJB823 is provided as SEQ ID NO: 15. The intracellular hydrocarbonproducts of E. coli K-12 EPI400™ (Epicentre) harboring pJB823, JCC1076,were compared to those of EPI400™ harboring pJB5, the control strainJCC9a, by gas chromatography-mass spectrometry (GC-MS). Clonal culturesof JCC9a and JCC1076 were grown overnight at 37° C. in Luria Broth (LB)containing 2% glucose, 100 μg/ml carbenicillin, 50 μg/ml spectinomycin,50 μg/ml streptomycin, and 1× CopyCutter Induction Solution (Epicentre).For each strain, 15 ml of saturated culture was collected bycentrifugation. Cell pellets were washed thoroughly by three cycles ofresuspension in Milli-Q water and microcentrifugation, and then dewettedas much as possible by three cycles of microcentrifugation andaspiration. Cell pellets were then extracted by vortexing for fiveminutes in 0.8 ml acetone containing 100 μg/ml butylated hydroxytoluene(BHT; a general antioxidant) and 100 μg/ml ethyl arachidate (EA; aninternal reporter of extraction efficiency). Cell debris was pelleted bycentrifugation, and 700 μl extractant was pipetted into a GC vial. TheseJCC9a and JCC1067 acetone samples, along with authentic standards, werethen analyzed by GC-MS.

The gas chromatograph was an Agilent 7890A GC equipped with a 5975Celectron-impact mass spectrometer. Liquid samples (1.0 μl) were injectedinto the GC with a 7683 automatic liquid sampler equipped with a 10 μlsyringe. The GC inlet temperature was 290° C. and split-less injectionwas used. The capillary column was an Agilent HP-5MS (30 m×0.25 mm×0.25μm). The carrier gas was helium at a flow rate of 1.0 ml/min. The GCoven temperature program was 50° C., hold 1 min/10° C. per min to 290°C./hold 9 min. The GC-MS interface temperature was 290° C. The MS sourcetemperature was 230° C., and the quadrapole temperature was 150° C. Themass range was 25-600 amu. MS fragmentation spectra were matched againstthe NIST MS database, 2008 version.

Peaks present in the total-ion GC-MS chromatograms were chemicallyassigned in one of two ways. In the first, assignment was done byensuring that both the retention time and the fragmentation massspectrum corresponded to the retention time and fragmentation massspectrum, respectively, of an authentic standard—this is referred to as“Method 1”, and is essentially unambiguous. In the absence of authenticstandards, only a tentative chemical assignment can be reached; this wasdone by collectively integrating the following data for the peak inquestion: (i) the structure of the fragmentation spectrum, especiallywith regard to the weight of the molecular ion, and to the degree towhich it resembled a hydrocarbon-characteristic “envelope” massspectrum, (ii) the retention time, especially with regard to itsqualitative compatibility with the assigned compound, e.g.,cis-unsaturated n-alkenes elute slightly before their saturated n-alkanecounterparts, and (iii) the likelihood that the assigned compound ischemically compatible with the operation of the AAR-ADM and relatedpathways in the host organism in question, e.g., fatty aldehydesgenerated by AAR are expected to be converted to the corresponding fattyalcohols by host dehydrogenases in E. coli if they are not acted uponsufficiently quickly by ADM. This second approach to peak assignment isreferred to as “Method 2”. In the total-ion GC-MS chromatogram in FIG.2, as well as in all such chromatograms in subsequent figures, peakschemically assigned by Method 1 are labeled in regular font, whereasthose assigned by Method 2 are labeled in italic font.

Total ion chromatograms (TICs) of JCC9a and JCC1076 acetone cell pelletextractants are shown in FIG. 2. The TICs of C₈-C₂₀ n-alkane authenticstandards (Sigma 04070), as well as 1-tetradecanol (Sigma 185388) plus1-hexadecanol (Sigma 258741) plus 1-octadecanol (Sigma 258768), are alsoshown. Hydrocarbons identified in JCC1076, but not in control strainJCC9a, are detailed in Table 3. These hydrocarbons are n-pentadecane(1), 1-tetradecanol (1), n-heptadecene (2), n-heptadecane (1), and1-hexadecanol (1), where the number in parentheses indicates the GC-MSpeak assignment method. MS fragmentation spectra of the Method 1 peaksare shown in FIG. 3, plotted against their respective library hits.

TABLE 3 Hydrocarbons detected by GC-MS in acetone cell pelletextractants of JCC1076 but not JCC9a, in increasing order of retentiontime. GC-MS Candidate Compound JCC9a JCC1076 Peak Assigment isomern-pentadecane − + Method 1 1-tetradecanol − + Method 1 n-heptadecene − +Method 2 cis-7- (envelope-type heptadecene MS with molecular ion mass238) n-pentadecane − + Method 1 1-hexadecanol − + Method 1 “−” notdetected; “+” detected.

The formation of these five products is consistent with both theexpected incomplete operation, i.e., acyl-ACP→fatty aldehyde→fattyalcohol, and expected complete operation, i.e., acyl-ACP→fattyaldehyde→alkane/alkene, of the AAR-ADM pathway in E. coli, whose majorstraight-chain acyl-ACPs include 12:0, 14:0, 16:0, 18:0, 16:1Δ9cis, and18:1Δ11cis acyl groups (Heipieper H J (2005); Appl Environ Microbiol71:3388). Assuming that n-heptadecene (2) is derived 18:1Δ11cis-ACP, itwould correspond to cis-7-heptadecene. Indeed, an n-heptadecene isomerwas identified as the highest-confidence MS fragmentation library hit atthat retention time, with the expected molecular ion of molecular weight238; also, as expected, it elutes slightly before n-heptadecane.

Example 3 Production of n-Alkanes, n-Alkenes, and Fatty Alcohols inEscherichia coli B through Heterologous Expression of Synechococcuselongatus PCC7942 SYNPCC7942_(—)1593 (adm) and SYNPCC7942_(—)1594 (aar)

The natural SYNPCC7942_(—)1593-SYNPCC7942_(—)1594 operonic sequence wasexcised from pJB823 using NdeI and EcoRI, and cloned into the commercialexpression vector pCDFDuet™-1 (Novagen) cut with via NdeI and Mfel. Theresulting plasmid was denoted pJB855 (SEQ ID NO: 16). This constructplaced the SYNPCC7942_(—)1593-SYNPCC7942_(—)1594 operon under thetranscriptional control of the inducible T7lacO promoter.

The intracellular hydrocarbon products of E. coli BL21(DE3) (Novagen)harboring pJB855, JCC1113, were compared to those of E. coli BL21(DE3)harboring pCDFDuet™-1, the control strain JCC114, by gaschromatography-mass spectrometry (GC-MS). Starter clonal cultures ofJCC1114 and JCC1113 were grown overnight at 37° C. in M9 minimal mediumsupplemented with 6 mg/l FeSO₄.7H₂O, 50 μg/ml spectinomycin, and 2%glucose as carbon source; this medium is referred to M9fs. Each starterculture was used to inoculate a 32 ml culture of M9fs at an initialOD₆₀₀ of 0.1. Inoculated cultures were grown at 37° C. at 300 rpm untilan OD₆₀₀ of 0.4 has been reached, at which point IPTG was added to afinal concentration of 1 mM. After addition of inducer, cultures weregrown under the same conditions for an additional 17 hours. For eachstrain, 12 ml of saturated culture was then collected by centrifugation.Cell pellets were washed thoroughly by 3 cycles of resuspension inMilli-Q water and microcentrifugation, and then dewetted as much aspossible by 3 cycles of microcentrifugation and aspiration. Cell pelletswere then extracted by vortexing for 5 minutes in 0.7 ml acetonecontaining 20 μg/ml BHT and 20 μg/ml EA. Cell debris was pelleted bycentrifugation, and 600 μl supernatant was pipetted into a GC vial.These JCC1114 and JCC1113 samples, along with authentic standards, werethen analyzed by GC-MS as described in Example 2. The TICs of JCC1114and JCC1113 acetone cell pellet extractants are shown in FIG. 4;n-alkane and 1-alkanol standards are as in Example 2. Hydrocarbonsidentified in JCC1113, but not in control strain JCC1114, are detailedin Table 4.

TABLE 4 Hydrocarbons detected by GC-MS in acetone cell pelletextractants of JCC1113 but not JCC1114 in increasing order of retentiontime. GC-MS Peak Candidate Compound JCC1114 JCC1113 Assigment isomern-tridecane − + Method 1 n-tetradecane − + Method 1 n-pentadecene − +Method 2 cis-7- (envelope-type pentadecene MS with molecular ion mass210) 1-dodecanol − + Method 2 n-pentadecane − + Method 1 n-hexadecene− + Method 2 cis-8- (envelope-type hexadecene MS with molecular ion mass224) n-hexadecane − + Method 1 1-tetradecanol − + Method 1 n-heptadecene− + Method 2 cis-7- (envelope-type heptadecene MS with molecular ionmass 238) n-heptadecane − + Method 1 1-pentadecanol − + Method 21-hexadecenol − + Method 2 cis-9- hexadecen- 1-ol 1-hexadecanol − +Method 1 1-octadecenol − + Method 2 cis-11- (envelope-type octadecen- MSwith 1-ol molecular ion mass 250) “−” not detected; “+” detected.

These hydrocarbons are n-tridecane (1), n-tetradecane (1), n-pentadecene(2), 1-dodecanol (2), n-pentadecane (1), n-hexadecene (2), n-hexadecane(1), 1-tetradecanol (1), n-heptadecene (2), n-heptadecane (1),1-pentadecanol (2), 1-hexadecenol (2), 1-hexadecanol (1), and1-octadecenol (2), where the number in parentheses indicates the GC-MSpeak assignment method. MS fragmentation spectra of Method 1 peaks areshown in FIG. 5, plotted against their respective library hits. Themajor products were n-pentadecane and n-heptadecene.

The formation of these fourteen products is consistent with both theexpected incomplete operation, i.e., acyl-ACP→>fatty aldehyde→fattyalcohol, and expected complete operation, i.e., acyl-ACP→fattyaldehyde→alkane/alkene, of the Aar-Adm pathway in E. coli, whose majorstraight-chain acyl-ACPs include 12:0, 14:0, 16:0, 18:0, 16:1Δ9cis, and18:1Δ11cis acyl groups (Heipieper H J (2005). Adaptation of Escherichiacoli to Ethanol on the Level of Membrane Fatty Acid Composition. ApplEnviron Microbiol 71:3388). Assuming that n-pentadecene (2) is derived16:1Δ9cis-ACP, it would correspond to cis-7-pentadecene. Indeed, ann-pentadecene isomer was identified as the highest-confidence MSfragmentation library hit at that retention time, with the expectedmolecular ion of molecular weight 210; also, as expected, it elutesslightly before n-pentadecane. With respect to 1-dodecanol (2), asufficiently clean fragmentation spectrum could not be obtained for thatpeak due to the overlapping, much larger n-pentadecane (1) peak. Itspresence, however, is consistent with the existence of 12:0-ACP in E.coli, and its retention time is exactly that extrapolated from therelationship between 1-alkanol carbon number and observed retentiontime, for the 1-tetradecanol, 1-hexadecanol, and 1-octadecanol authenticstandards that were run. Assuming that n-hexadecene (2) is derived fromthe trace-level unsaturated 17:1Δ9cis acyl group expected in the E. coliacyl-ACP population due to rare acyl chain initiation with propionyl-CoAas opposed to malonyl-CoA, it would correspond to cis-8-hexadecene.Indeed, an n-hexadecene isomer was identified as the highest-confidenceMS fragmentation library hit at that retention time, with the expectedmolecular ion of molecular weight 224; also, as expected, it elutesslightly before n-hexadecane. Assuming that n-heptadecene (2) is derived18:1Δ11cis-ACP, it would correspond to cis-7-heptadecene. Indeed, ann-heptadecene isomer was identified as the highest-confidence MSfragmentation library hit at that retention time, with the expectedmolecular ion of molecular weight 238; also, as expected, it elutesslightly before n-heptadecane. With respect to 1-pentadecanol (2), asufficiently clean fragmentation spectrum could not be obtained for thatpeak due to its low abundance. Its presence, however, is consistent withthe existence of trace-level 15:0 acyl group expected in the E. coliacyl-ACP population due to rare acyl chain initiation with propionyl-CoAas opposed to malonyl-CoA, and its retention time is exactly thatinterpolated from the relationship between 1-alkanol carbon number andobserved retention time, for the 1-tetradecanol, 1-hexadecanol, and1-octadecanol authentic standards that were run. In addition,1-pentadecanol was identified as the highest-confidence MS fragmentationlibrary hit at that retention time in acetone extracts of JCC1170, aBL21(DE3) derivative that expresses Aar without Adm (see Example 4).With respect to 1-hexadecenol (2), a sufficiently clean fragmentationspectrum could not be obtained for that peak due to its low abundance;however, assuming that it is derived 16:1Δ9cis-ACP, it would correspondto cis-9-hexadecen-1-ol. Also, as expected, it elutes slightly before1-hexadecanol. Finally, assuming that n-octadecenol (2) is derived18:1Δ9cis-ACP, it would correspond to cis-11-octadecen-1-ol. Indeed, ann-octadecen-1-ol isomer was identified as the highest-confidence MSfragmentation library hit at that retention time, with the expectedmolecular ion of molecular weight 250; also, as expected, it elutesslightly before 1-octadecanol.

Example 4 Production of Fatty Alcohols in Escherichia coli B throughHeterologous Expression of Synechococcus elongatus SYNPCC7942_(—)1594(aar) without Co-Expression of SYNPCC7942_(—)1593 (adm)

In order to test the hypothesis that both AAR and ADM are required foralkane biosynthesis, as well as the prediction that expression of AARalone should result in the production of fatty alcohols only in E. coli(due to non-specific dehydrogenation of the fatty aldehydes generated),expression constructs containing just SYNPCC7942_(—)1593 (ADM) and justSYNPCC7942_(—)1594 (AAR), were created. Accordingly, theSYNPCC7942_(—)1593 and SYNPCC7942_(—)1594 coding sequences wereindividually PCR-amplified and cloned via NdeI and Mfel into thecommercial expression vector pCDFDuet™-1 (Novagen). The resultingplasmids were denoted pJB881 (SYNPCC7942_(—)1593 only) and pJB882(SYNPCC7942_(—)1594 only); in each construct, the coding sequence wasplaced under the transcriptional control of the inducible T7lacOpromoter.

The intracellular hydrocarbon products of E. coli BL21(DE3) (Novagen)harboring pJB881, JCC1169, and of E. coli BL21(DE3) (Novagen) harboringpJB882, JCC1170, were compared to those of E. coli BL21(DE3) harboringpCDFDuet™-1, the negative control strain JCC114, as well as to thepositive control SYNPCC7942_(—)1593-SYNPCC7942_(—)1594 strain JCC1113(Example 3), by gas chromatography-mass spectrometry (GC-MS). Clonalcultures of JCC1169, JCC1170, JCC1114, and JCC1113 were grown,extracted, and analyzed by GC-MS as described in Example 3, with thefollowing exception: the JCC1170 culture was grown overnight in M9fsmedium without IPTG, because the culture did not grow if IPTG was added.Presumably, this was due to the toxic over-accumulation of fattyalcohols that occurred even in the absence of inducer.

The TICs of JCC1169, JCC1170, JCC1114, and JCC1113 acetone cell pelletextractants are shown in FIG. 6; n-alkane and 1-alkanol standard traceshave been omitted. Hydrocarbons identified in JCC1170, but not incontrol strain JCC1114, are detailed in Table 5.

TABLE 5 Hydrocarbons detected by GC-MS in acetone cell pelletextractants of JCC1170 but not JCC1114 in increasing order of retentiontime. GC-MS Candidate Compound JCC1114 JCC1170 Peak Assigment isomer1-tetradecanol − + Method 1 1-pentadecanol − + Method 2 (envelope- typeMS with molecular ion mass 182) 1-hexadecenol − + Method 2 cis-9-(envelope- hexadecen- type MS 1-ol with molecular ion mass 222)1-hexadecanol − + Method 1 1-octadecenol − + Method 2 cis-11- (envelope-octadecen- type MS 1-ol with molecular ion mass 250) “−” not detected;“+” detected.

These hydrocarbons are 1-tetradecanol (1), 1-pentadecanol (2),1-hexadecenol (2), 1-hexadecanol (1), and 1-ocadecenol (2), where thenumber in parentheses indicates the GC-MS peak assignment method. MSfragmentation spectra of Method 1 peaks are shown in FIG. 7, plottedagainst their respective library hits. No hydrocarbons were identifiedin JCC1169, whose trace was indistinguishable from that of JCC1114, asexpected owing to absence of fatty aldehyde substrate generation by AAR.

The lack of production of alkanes, alkenes, and fatty alkanols inJCC1169, the production of only fatty alcohols in JCC1170, and theproduction of alkanes, alkenes, and fatty alkanols in JCC1113 (asdiscussed in Example 3) are all consistent with the proposed mechanismof alkane biosynthesis by AAR and ADM in E. coli. Thus, the formation ofthe five fatty alcohols in JCC1170 is consistent with only AAR beingactive, and active on the known straight-chain acyl-ACPs (see Example3). With respect to 1-pentadecanol (2), its presence is consistent withthe existence of trace-level 15:0 acyl group expected in the E. coliacyl-ACP population due to rare acyl chain initiation with propionyl-CoAas opposed to malonyl-CoA and its retention time is exactly thatinterpolated from the relationship between 1-alkanol carbon number andobserved retention time, for the 1-tetradecanol, 1-hexadecanol, and1-octadecanol authentic standards that were run. Most importantly, the1-pentadecanol (2) peak exhibits an envelope-type fragmentation massspectrum, with the expected molecular ion of molecular weight 182.Unlike in the case of JCC1113, a clean fragmentation spectrum from thecandidate 1-hexadecenol peak could now be obtained due to increasedabundance. The top library hit was a 1-hexadecenol with the expectedmolecular ion of molecular weight 222. Assuming that it is derived from16:1Δ9cis hexadecenyl-ACP, the isomeric assignment would becis-9-hexadecen-1-ol; also, as expected, it elutes slightly before1-hexadecanol. Assuming that n-octadecenol (2) is derived18:11Δ9cis-ACP, it would correspond to cis-11-octadecen-1-ol. Indeed, ann-octadecen-1-ol isomer was identified as the highest-confidence MSfragmentation library hit at that retention time, with the expectedmolecular ion of molecular weight 250; also, as expected, it elutesslightly before 1-octadecanol. There is also an unidentified side peakin JCC1170 that elutes in the tail of 1-hexadecenol and whosefragmentation mass spectrum was not sufficiently clean to enablepossible identification. It is hypothesized that this could be theprimary C₁₈ aldehyde product expected of AAR-only activity in E. coli,i.e., cis-11-octadecenal.

Example 5 Production of n-Alkanes, n-Alkenes, and Fatty Alcohol inSynechococcus sp. PCC 7002 through Heterologous Expression ofSynechococcus elongatus PCC7942 SYNPCC7942_(—)1593 (adm) andSYNPCC7942_(—)1594 (aar)

In order to test whether heterologous expression of AAR and ADM wouldlead to the desired alkane biosynthesis in a cyanobacterial host, theSYNPCC7942_(—)1593-SYNPCC7942_(—)1594 operon was expressed inSynechococcus sp. PCC 7002 (JCC138). Accordingly, plasmid pJB823 wastransformed into JCC138, generating strain JCC1160. The sequence andannotation of this plasmid is provided as SEQ ID NO: 15, and describedin Example 2. In this construct, theSYNPCC7942_(—)1593-SYNPCC7942_(—)1594 operon is placed under thetranscriptional control of the constitutive aphII promoter. 500 basepair upstream and downstream homology regions direct homologousrecombinational integration into the native high-copy pAQ1 plasmid ofJCC138, and an aadA gene permits selection of transformants by virtue oftheir resistance to spectinomycin.

To test the effect of potentially stronger promoters, constructsdirectly analogous to pJB823 were also generated that substituted theaphII promoter with the following: the promoter of cro from lambda phage(PcI), the promoter of cpcB from Synechocystis sp. PCC 6803 (PcpcB), thetrc promoter along with an upstream copy of a promoter-lacI cassette(PlacI-trc), the synthetic EM7 promoter (PEM7). Promoters were exchangedvia the NotI and NdeI sites flanking the promoter upstream of theSYNPCC7942_(—)1593-SYNPCC7942_(—)1594 operon in the pJB823 vector. Thecorresponding final plasmids were as follows: pJB886 (PcI), pJB887(PcpcB), pJB889 (PlacI-trc), pJB888 (PEM7), and pJB823 (Paphll). Thesesequences of pJB886, pJB887, pJB889, and pJB888 are identical to thesequence of pJB823 except in the region between the NotI and NdeI sites,where they differ according to the promoter used. The sequences of thedifferent promoter regions are provided as SEQ ID NO: 19 (PcI), SEQ IDNO: 20 (PcpcB), SEQ ID NO: 21 (PlacI-trc), and SEQ ID NO: 22 (PEM7). Thesequence of the PaphII promoter is presented in SEQ ID NO: 15.

pJB886, pJB887, pJB889, pJB888, pJB823, as well as pJB5 (the empty pAQ1targeting vector that entirely lacked theSYNPCC7942_(—)1593-SYNPCC7942_(—)1594 operonic sequence) were naturallytransformed into JCC138 using a standard cyanobacterial transformationprotocol, generating strains JCC1221 (PcI), JCC1220 (PcpcB), JCC1160b(PlacI-trc), JCC1160a (PEM7), JCC1160 (PaphII), and JCC879 (pJB5),respectively. Briefly, 5-10 μg of plasmid DNA was added to 1 ml of neatJCC138 culture that had been grown to an OD₇₃₀ of approximately 1.0. Thecell-DNA mixture was incubated at 37° C. for 4 hours in the dark withgentle mixing, plated onto A+ plates, and incubated in a photoincubator(Percival) for 24 hours, at which point spectinomycin was underlaid to afinal concentration of 50 μg/ml. Spectinomycin-resistant coloniesappeared after 5-8 days of further incubation under 24 hr-lightconditions (˜100 μmol photons m⁻² s⁻¹). Following one round of colonypurification on A+ plates supplemented with 100 μg/ml spectinomycin,single colonies of each of the six transformed strains were grown intest-tubes for 4-8 days at 37° C. at 150 rpm in 3% CO₂-enriched air at˜100 μmol photons m⁻² s⁻¹ in a Multitron II (Infors) shakingphotoincubator. The growth medium used for liquid culture was A+ with200 μg/ml spectinomycin.

In order to compare the intracellular hydrocarbon products of strainsJCC1221, JCC1220, JCC1160b, JCC1160a, JCC1160, and JCC879, 24 OD₇₃₀-mlworth of cells (˜2.4×10⁹ cells) of each strain was collected from theaforementioned test-tube cultures by centrifugation. Cell pellets werewashed thoroughly by 3 cycles of resuspension in Milli-Q water andmicrocentrifugation, and then dewetted as much as possible by 3 cyclesof microcentrifugation and aspiration. Cell pellets were then extractedby vortexing for 5 minutes in 0.7 ml acetone containing 20 μg/ml BHT and20 μg/ml EA. Cell debris was pelleted by centrifugation, and 600 μlsupernatant was pipetted into a GC vial. The six extractants, along withauthentic standards, were then analyzed by GC-MS as described in Example2.

The TICs of JCC1221, JCC1220, JCC1160b, JCC1160a, JCC1160, and JCC879acetone cell pellet extractants are shown in FIG. 8; n-alkane and1-alkanol standards are as in Example 2. Consistent with a range ofpromoter strengths, and with function of the AAR-ADM pathway, there wasa range of hydrocarbon accumulation, the order of accumulation beingPcI>PcpcB>PlacI-trc>PEM7>PaphII (FIG. 8A).

In JCC1160, approximately 0.2% of dry cell weight was found as n-alkanesand n-alkan-1-ol (excluding n-nonadec-1-ene). Of this 0.2%,approximately three-quarters corresponded to n-alkanes, primary productsbeing n-heptadecane and n-pentadecane. These hydrocarbons were notdetected in JCC879. The data are summarized in Table 6A.

TABLE 6A Hydrocarbons detected in acetone extracts of JCC1160 andJCC879. Approximate % of dry cell weight Compound JCC879 JCC1160n-pentadecane not detected 0.024% n-hexadecane nd 0.004% n-heptadecanend 0.110% n-octadecan-1-ol nd 0.043% Total 0.181% % of products that aren-alkanes 76%

The highest accumulator was JCC1221 (PcI). Hydrocarbons identified inJCC1221, but not in control strain JCC879, are detailed in Table 6B,Table 6C and FIG. 8B. These hydrocarbons are n-tridecane (1),n-tetradecane (1), n-pentadecene (2), n-pentadecane (1), n-hexadecane(1), n-heptadec-di-ene (2), three isomers of n-heptadecene (2),n-heptadecane (1), and 1-ocadecanol (1), where the number in parenthesesindicates the GC-MS peak assignment method.

TABLE 6B n-Alkanes quantitated in acetone extract of JCC1221 Compound %of JCC1221 dry cell weight n-tridecane 0.001%  n-tetradecane 0.0064%n-pentadecane 0.40%  n-hexadecane 0.040%  n-heptadecane 1.2%   Total1.67% 

MS fragmentation spectra of Method 1 peaks are shown in FIG. 9, plottedagainst their respective library hits. The only alkanes/alkenes observedin JCC879 were 1-nonadecene and a smaller amount of nonadec-di-ene,alkenes that are known to be naturally synthesized by JCC138 (Winters Ket al. (1969) Science 163:467-468). The major products observed inJCC1221 were n-pentadecane (˜25%) and n-heptadecane (˜75%); all otherswere in relatively trace levels.

The formation of n-pentadecane and n-heptadecane in JCC1221, as well asthe nine other trace hydrocarbon products, is consistent with thevirtually complete operation of the ADM-AAR pathway in JCC138, i.e.,16:0 hexadecyl-ACP→n-hexadecanal→n-pentadecane and 18:0octadecyl-ACP→n-octadecanal→n-heptadecane. Indeed it is known that themajor acyl-ACP species in this organism are C_(16:0) and C_(18:0)(Murata N et al. (1992) Plant Cell Physiol 33:933-941). Relatively muchless fatty alcohol is produced relative to AAR-ADM expression in E. coli(Example 3), as expected given the presence in JCC138 of acyanobacterial ferredoxin/ferredoxin-NADPH reductase system that canregenerate the di-iron active site of ADM, thereby preventing theaccumulation of hexadecanal and octadecanal that could in turn benon-specifically dehydrogenated to the corresponding 1-alkanols. Thus,in JCC1221, only a very small 1-octadecanol (1) peak is observed (FIG.8).

The other trace hydrocarbons seen in JCC1221 are believed to beunsaturated isomers of n-pentadecane and n-heptadecane (Table 6C). It ishypothesized that all these alkenes are generated by desaturation eventsfollowing the production of the corresponding alkanes by theSYNPCC7942_(—)1593 Adm. This contrasts with the situation in E. coli,where double bonds are introduced into the growing acyl chain while itis linked to the acyl carrier protein (Example 3). JCC138 is known tohave a variety of position-specific acyl-lipid desaturases that, whilenominally active only on fatty acids esterified to glycerolipids, couldpotentially act on otherwise unreactive alkanes producednonphysiologically by the action of AAR and ADM. JCC138 desaturases,i.e., DesA, DesB, and DesC, introduce cis double bonds at the Δ9, Δ12,and Δ15 positions of C₁₈ acyl chains, and at the Δ9 and Δ12 positions ofC₁₆ acyl chains (Murata N and Wada H (1995) Biochem J. 308:1-8). Thecandidate n-pentadecene peak is believed to be cis-4-pentadecene (Table6C).

Assuming also that heptadecane could also serve as a substrate forJCC138 desaturases, and that it would be desaturated at positionsanalogous to the Δ9, Δ12, and Δ15 of the C₁₈ acyl moiety, there are fourtheoretically possible mono-unsaturated isomers: cis-3-heptadecene,cis-6-heptadecene, cis-8-heptadecene, and cis-9-heptadecene. Theseisomers do not include the single n-heptadecene species nominallyobserved in E. coli, cis-7-heptadecene (Example 2). It is believed thatthe three peaks closest to the n-heptadecane peak—denoted by subscripts1, 2, and 3 in Table 6C and FIG. 8B—encompass at least three of thesefour mono-unsaturated heptadecane isomers. Consistent with this,n-heptadecene₂ and n-heptadecene₃ peaks have the expected molecular ionsof mass 238 in their envelope-type fragmentation spectra. There are manyisomeric possibilities, accordingly, for the putativecis,cis-heptadec-di-ene peak, which has an envelope-type fragmentationspectrum with the expected molecular ions of mass 236. As expected, allputative heptadecene species elute slightly before n-heptadecane.

TABLE 6C Alkane and alkenes detected by GC-MS in acetone cell pelletextractants of JCC1221 but not JCC879 in increasing order of retentiontime. GC-MS Peak Candidate Compound JCC879 JCC1221 Assigment isomern-tridecane − + Method 1 n-tetradecane − + Method 1 n-pentadecene − +Method 2 cis-4- pentadecene n-pentadecane − + Method 1 n-hexadecane − +Method 1 n-heptadec- − + Method 2 cis,cis- di-ene (envelope-typeheptadec-di-ene MS with molecular ion mass 236) n-heptadecene₃ − +Method 2 cis-[3/6/8/9]- (envelope-type heptadecene MS with molecular ionmass 238) n-heptadecene₂ − + Method 2 cis-[3/6/8/9]- (envelope-typeheptadecene MS with molecular ion mass 238) n-heptadecene₁ − + Method 2cis-[3/6/8/9]- heptadecene n-heptadecane − + Method 1 1-octadecanol − +Method 1 “−”, not detected; “+”, detected.

Example 6 Intracellular Accumulation of n-Alkanes to Up to 5% of DryCell Weight in Synechococcus sp. PCC 7002 through HeterologousExpression of Synechococcus elongatus PCC7942 SYNPCC7942_(—)1593 (adm)and SYNPCC7942_(—)1594 (aar)

In order to quantitate more accurately the level of intracellularaccumulation of n-alkane products in the alkanogen JCC1221 (Example 5),the levels of n-pentadecane and n-heptadecane, as well as the relativelytrace products n-tetradecane and n-hexadecane, were quantified withrespect to dry cell weight (DCW). Based on the hypothesis that theextent of n-alkane production could correlate positively with the levelof SYNPCC7942_(—)1593-SYNPCC7942_(—)1594 operon expression, theDCW-normalized n-alkane levels of JCC1221 were determined as a functionof the spectinomycin concentration of the growth medium. The rationalewas that the higher the spectinomycin selective pressure, the higher therelative copy number of pAQ1, and the more copies of the aadA-linkedSYNPCC7942_(—)1593-SYNPCC7942_(—)1594 operon.

A clonal starter culture of JCC1221 was grown up in A+ mediumsupplemented with 100 μg/ml spectinomycin in for 7 days at 37° C. at 150rpm in 3% CO₂-enriched air at ˜100 μmol photons m⁻² s⁻¹ in a MultitronII (Infors) shaking photoincubator. At this point, this culture was usedto inoculate triplicate 30 ml JB2.1 medium (PCT/US2009/006516) flaskcultures supplemented with 100, 200, 300, 400, or 600 μg/mlspectinomycin. JB2.1 medium consists of 18.0 g/l sodium chloride, 5.0g/l magnesium sulfate heptahydrate, 4.0 g/l sodium nitrate, 1.0 g/lTris, 0.6 g/l potassium chloride, 0.3 g/l calcium chloride (anhydrous),0.2 g/l potassium phosphate monobasic, 34.3 mg/l boric acid, 29.4 mg/lEDTA (disodium salt dihydrate), 14.1 mg/l iron (III) citrate hydrate,4.3 mg/l manganese chloride tetrahydrate, 315.0 μg/l zinc chloride, 30.0μg/l molybdenum (VI) oxide, 12.2 μg/l cobalt (II) chloride hexahydrate,10.0 μg/l vitamin B₁₂, and 3.0 μg/l copper (II) sulfate pentahydrate.The 15 cultures were grown for 10 days at 37° C. at 150 rpm in 3%CO₂-enriched air at ˜100 μmol photons m⁻² s⁻¹ in a Multitron II (Infors)shaking photoincubator.

For each culture, 5-10 ml was used for dry cell weight determination. Todo so, a defined volume of culture—corresponding to approximately 20 mgDCW—was centrifuged to pellet the cells. Cells were transferred to apre-weighed eppendorf tube, and then washed by 2 cycles of resuspensionin Milli-Q water and microcentrifugation, and dewetted by 3 cycles ofmicrocentrifugation and aspiration. Wet cell pellets were frozen at −80°C. for two hours and then lyophilized overnight, at which point the tubecontaining the dry cell mass was weighed again such that the mass of thecell pellet could be calculated within ±0.1 mg. In addition, for eachculture, 0.3-0.8 ml was used for acetone extraction of the cell pelletfor GC analysis. To do so, a defined volume of culture—corresponding toapproximately 1.4 mg DCW—was microcentrifuged to pellet the cells. Cellswere then washed by 2 cycles of resuspension in Milli-Q water andmicrocentrifugation, and then dewetted by 4 cycles ofmicrocentrifugation and aspiration. Dewetted cell pellets were thenextracted by vortexing for 1 minute in 1.0 ml acetone containing 50μg/ml BHT and 160 μg/ml n-heptacosane internal standard (Sigma 51559).Cell debris was pelleted by centrifugation, and 700 μl supernatant waspipetted into a GC vial.

Concentrations of n-tetradecane, n-pentadecane, n-hexadecane, andn-heptadecane in the fifteen extractants were quantitated by gaschromatography/flame ionization detection (GC/FID). Unknown n-alkanepeak areas in biological samples were converted to concentrations vialinear calibration relationships determined between known n-tetradecane,n-pentadecane, n-hexadecane, and n-heptadecane authentic standardconcentrations and their corresponding GC-FID peak areas. Standards wereobtained from Sigma. GC-FID conditions were as follows. An Agilent 7890AGC/FID equipped with a 7683 series autosampler was used. 1 μl of eachsample was injected into the GC inlet (split 5:1, pressure: 20 psi,pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 ml/min) and aninlet temperature of 280° C. The column was a HP-5MS (Agilent, 30 m×0.25mm×0.25 μm) and the carrier gas was helium at a flow of 1.0 ml/min. TheGC oven temperature program was 50° C., hold one minute; 10° C./minincrease to 280° C.; hold ten minutes. n-Alkane production wascalculated as a percentage of the DCW extracted in acetone.

Consistent with scaling between pAQ1 selective pressure and the extentof intracellular n-alkane production in JCC1221, there was a roughlypositive relationship between the % n-alkanes with respect to DCW andspectinomycin concentration (FIG. 10). For all JCC1221 cultures,n-alkanes were ˜25% n-pentadecane and ˜75% n-heptadecane. The minimumn-alkane production was ˜1.8% of DCW at 100 μg/ml spectinomycin and 5.0%in one of the 600 μg/ml spectinomycin cultures.

Example 7 Production of n-Alkanes in Synechococcus sp. PCC 7002 throughHeterologous Expression of Prochlorococcus marinus MIT 9312PMT9312_(—)0532 (adm) and PMT9312_(—)0533 (aar)

This candidate Adm/Aar pair from Prochlorococcus marinus MIT9312 wasselected for functional testing by heterologous expression in JCC138because of the relatively low amino acid homology (>62%) of theseproteins to their Synechococcus elongatus PCC7942 counterparts,SYNPCC7942_(—)1593 and SYNPCC7942_(—)1594. Specifically, the 252-aminoacid protein PMT9312_(—)0532 exhibits only 62% amino acid identity withthe 232 amino acid protein SYNPCC7942_(—)1593, wherein amino acids33-246 of the former are aligned with amino acids 11-224 of the latter.The 347 amino acid protein PMT9312_(—)0533 exhibits only 61% amino acididentity with the 342 amino acid protein SYNPCC7942_(—)1594, whereinamino acids 1-337 of the former are aligned with amino acids 1-339 ofthe latter.

A codon- and restriction-site-optimized version of thePMT9312_(—)0532-PMT9312_(—)0533 operon was synthesized by DNA2.0 (MenloPark, Calif.), flanked by NdeI and EcoRI sites. The operon was clonedinto the pAQ1 homologous recombination vector pJB5 via NdeI and EcoRI,such that the PMT9312_(—)0532-PMT9312_(—)0533 operon was placed undertranscriptional control of the aphII promoter. The sequence of thepJB947 vector is provided as SEQ ID NO: 17.

pJB947 was transformed into JCC138 as described in Example 5, generatingstrain JCC1281. The hydrocarbon products of this strain were compared tothose of the negative control strain JCC879, corresponding to JCC138transformed with empty pJB5 (see Example 5). Eight OD₇₃₀-ml worth ofcells (˜8×10⁸ cells) of each strain was collected by centrifugation,having been grown in A+ medium supplemented with 200 μg/ml spectinomycinas described in Example 5. Cell pellets were washed thoroughly by 3cycles of resuspension in Milli-Q water and microcentrifugation, andthen dewetted as much as possible by 3 cycles of microcentrifugation andaspiration. Cell pellets were then extracted by vortexing for 5 minutesin 0.7 ml acetone containing 20 μg/ml BHT and 20 μg/ml EA. Cell debriswas pelleted by centrifugation, and 600 μl supernatant was pipetted intoa GC vial. Samples were analyzed by GC-MS as described in Example 5.

The TICs of JCC1281 and JCC879 acetone cell pellet extractants are shownin FIG. 11; n-alkane standards are as in Example 6. Hydrocarbonsidentified in JCC1281, but not in control strain JCC879, weren-pentadecane (1) and n-heptadecane (1), where the number in parenthesesindicates the GC-MS peak assignment method. MS fragmentation spectra ofMethod 1 peaks are shown in FIG. 12, plotted against their respectivelibrary hits (as noted in Example 5, the only alkanes/alkenes observedin JCC879 were 1-nonadecene and a smaller amount of nonadec-di-ene,alkenes that are known to be naturally synthesized by JCC138). Theamount of n-alkanes produced in JCC1281 is at least 0.1% dry cellweight, and at least 2-two times higher than the amount produced byJCC879. The ratio of n-pentadecane:n-heptadecane (˜40%:˜60%) in JCC1281was higher than that observed in JCC1221 (˜25%:˜75%), suggesting thatthe PMT9312_(—)0532 (ADM) and/or the PMT9312_(—)0533 (AAR) exhibithigher activity towards the C₁₆ substrates relative to C₁₈ substrates,compared to SYNPCC7942_(—)1593 (ADM) and/or SYNPCC7942_(—)1594 (AAR).

Example 8 Augmentation of Native n-Alkane Production inThermosynechococcus elongatus BP-1 by Overexpression of the Nativetll1313 (adm)-tll1312 (aar) Operon

Genes encoding Thermosynechococcus elongatus BP-1 tll1312 (AAR) andtll1313 (ADM) are incorporated into one or more plasmids (e.g., pJB5derivatives), comprising promoters of differing strength. The plasmidsare used to transform Thermosynechococcus elongatus BP-1. Overexpressionof the genes in the transformed cells are measured as will the amount ofn-alkanes, particularly heptadecane, produced by the transformed cells,in a manner similar to that described in Example 3. The n-alkanes andother carbon-based products of interest can also be isolated from thecell or cell culture, as needed.

Wild-type Thermosynechococcus elongatus BP-1, referred to as JCC3,naturally produces n-heptadecane as the major intracellular hydrocarbonproduct, with traces of n-hexadecane and n-pentadecane. These n-alkaneswere identified by GC-MS using Method 1; fragmentation spectra are shownin FIG. 13. Briefly, a colony of JCC3 was grown in B-HEPES medium to afinal OD₇₃₀ of ˜4, at which point 5 OD₇₃₀-ml worth of cells washarvested, extracted in acetone, and analyzed by GC-MS as detailed inExample 5.

In an effort to augment this n-alkane production, the nativetll1313-tll1312 operonic sequence from this organism was PCR-amplifiedand cloned into the Thermosynechococcus elongatus BP-1 chromosomalintegration vector pJB825. This construct places the tll1313-tll1312operon under the transcriptional control of the constitutive cIpromoter. The sequence of the resulting plasmid, pJB825t, is shown inSEQ ID NO:18.

pJB825 and pJB825t were naturally transformed into JCC3 using a standardcyanobacterial transformation protocol, generating strains JCC1084 andJCC1084t, respectively. Briefly, 25 μg of plasmid DNA was added to 0.5ml of concentrated JCC3 culture (OD₇₃₀ ˜100) that had originally beengrown to an OD₇₃₀ of approximately 1.0 in B-HEPES at 45° C. in 3%CO₂-enriched air at ˜100 μmol photons m⁻² s⁻¹ in a Multitron II (Infors)shaking photoincubator. The cell-DNA mixture was incubated at 37° C. for4 hours in the dark with gentle mixing, made up to 7 ml with freshB-HEPES medium, and then incubated under continuous light conditions(˜100 μmol photons m⁻² s⁻¹) for 20 hours at 45° C. at 150 rpm in 3%CO₂-enriched air at ˜100 μmol photons m⁻² s⁻¹ in a Multitron II (Infors)shaking photoincubator. At this point, cells were collected bycentrifugation and serial dilutions were mixed with molten top agar andplated on the surface of B-HEPES plates supplemented with 60 μg/mlkanamycin. Transformant colonies appeared in the top agar layer withinaround 7 days upon incubation in a photoincubator (Percival) in 1%CO₂-enriched air at continuous ˜100 μmol photons m⁻² s⁻¹ irradiance.Single colonies of JCC1084 and JCC1084t were then grown up in triplicateto an OD₇₃₀ of ˜6 in B-HEPES/60 μg/ml kanamycin liquid culture, andtheir intracellular hydrocarbon products quantitated by GC-FID.

3.5 OD₇₃₀-ml worth of cells (˜3.5×10⁸ cells) of each replicate cultureof each strain was collected by centrifugation. Cell pellets were washedthoroughly by 3 cycles of resuspension in Milli-Q water andmicrocentrifugation, and then dewetted as much as possible by 3 cyclesof microcentrifugation and aspiration. Cell pellets were then extractedby vortexing for 1 minutes in 0.7 ml acetone containing 20 μg/ml BHT and20 μg/ml n-heptacosane. Cell debris was pelleted by centrifugation, and600 μl supernatant was pipetted into a GC vial. The two extractants,along with authentic C₈-C₂₀ n-alkane authentic standards (Sigma 04070),were then analyzed by GC coupled with flame ionization detection (FID)as described in Example 6. Quantitation of n-pentadecane, n-hexadecane,and n-heptadecane by GC-FID, and dry cell weights were taken asdescribed in Example 6.

Consistent with increased expression of tll1313-tll1312 in JCC1084trelative to the control strain JCC1084, n-pentadecane, n-hexadecane, andn-heptadecane were ˜500%, ˜100%, and ˜100% higher, respectively, inJCC1084t relative to their % DCW levels in JCC1084 (FIG. 14). The totaln-alkane concentration in both strains was less than 1%. The n-alkaneconcentration in JCC1084t was at least 0.62% and at least twice as muchn-alkane was produced relative to JCC1084.

Example 9 Comparison of Intracellular Hydrocarbon Products of JCC1113 (aDerivative of E. coli) and JCC1221 (a Derivative of Synechococcus sp.PCC 7002), both Strains Heterologously expressing Synechococcuselongatus SYNPCC7942_(—)1593 (adm) and SYNPCC7942_(—)1594 (aar)

GC-MS TICs of JCC1113 and JCC1221 acetone cell pellet extractants areshown in FIG. 15, along with the TIC of C₈-C₂₀ n-alkane authenticstandards (Sigma 04070). These two strains are derived from E. coliBL21(DE3) and Synechococcus sp. PCC7002, respectively, and are describedin detail in Examples 3 and 5, respectively. JCC1113 synthesizespredominantly n-heptadecene and n-pentadecane, whereas JCC1221synthesizes predominantly n-heptadecane and n-pentadecane. This figurevisually emphasizes the different retention times of the n-heptadeceneisomer produced in JCC1113 and n-heptadecane produced in JCC1221.

Example 10 Production of Hydrocarbons in Yeast The methods of theinvention can be performed in a number of lower eukaryotes such asSaccharomyces cerevisiae, Trichoderma reesei, Aspergillus nidulans andPichia pastoris. Engineering such organisms may include optimization ofgenes for efficient transcription and/or translation of the encodedprotein. For instance, because the ADM and AAR genes introduced into afungal host are of cyanobacterial origin, it may be necessary tooptimize the base pair composition. This includes codon optimization toensure that the cellular pools of tRNA are sufficient. The foreign genes(ORFs) may contain motifs detrimental to completetranscription/translation in the fungal host and, thus, may requiresubstitution to more amenable sequences. The expression of eachintroduced protein can be followed both at the transcriptional andtranslational stages by well known Northern and Western blottingtechniques, respectively.

Use of various yeast expression vectors including genes encodingactivities which promote the ADM or AAR pathways, a promoter, aterminator, a selectable marker and targeting flanking regions. Suchpromoters, terminators, selectable markers and flanking regions arereadily available in the art. In a preferred embodiment, the promoter ineach case is selected to provide optimal expression of the proteinencoded by that particular ORF to allow sufficient catalysis of thedesired enzymatic reaction. This step requires choosing a promoter thatis either constitutive or inducible, and provides regulated levels oftranscription. In another embodiment, the terminator selected enablessufficient termination of transcription. In yet another embodiment, theselectable/counterselectable markers used are unique to each ORF toenable the subsequent selection of a fungal strain that contains aspecific combination of the ORFs to be introduced. In a furtherembodiment, the locus to which relevant plasmid construct (encodingpromoter, ORF and terminator) is localized, is determined by the choiceof flanking region.

The engineered strains can be transformed with a range of differentgenes for production of carbon-based products of interest, and thesegenes are stably integrated to ensure that the desired activity ismaintained throughout the fermentation process. Various combinations ofenzyme activities can be engineered into the fungal host such as theADM, ADR pathways while undesired pathways are attenuated or knockedout.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention. Allpublications, patents and other references mentioned herein are herebyincorporated by reference in their entirety.

1. A method for producing hydrocarbons, comprising: (i) culturing anengineered cyanobacterium in a culture medium, wherein said engineeredcyanobacterium comprises a recombinant acyl ACP reductase (AAR) enzymeand a recombinant alkanal decarboxylative monooxygenase (ADM) enzyme;and (ii) exposing said engineered cyanobacterium to light and carbondioxide, wherein said exposure results in the conversion of said carbondioxide by said engineered cynanobacterium into n-alkanes, wherein atleast one of said n-alkanes is selected from the group consisting ofn-tridecane, n-tetradecane, n-pentadecane, n-hexadecane, andn-heptadecane, and wherein the amount of said n-alkanes produced isbetween 0.1% and 5% dry cell weight and at least two times the amountproduced by an otherwise identical cyanobacterium, cultured underidentical conditions, but lacking said recombinant AAR and ADM enzymes.2. The method of claim 1, wherein at least one of said recombinantenzymes is heterologous with respect to said engineered cyanobacterium.3. The method of claim 1, wherein said engineered cyanobacterium furtherproduces at least one n-alkene or n-alkanol.
 4. The method of claim 3,wherein said engineered cyanobacterium produces at least one n-alkene orn-alkanol selected from the group consisting of n-pentadecene,n-heptadecene, and 1-octadecanol.
 5. The method of claim 3, wherein saidn-alkanes comprise predominantly n-heptadecane, n-pentadecane or acombination thereof.
 6. The method of claim 3, further comprisingisolating at least one n-alkane, n-alkene or n-alkanol from saidengineered cyanobacterium or said culture medium.
 7. The method of claim1, wherein said enzymes are encoded by a plasmid.
 8. The method of claim1 wherein said enzymes are encoded by recombinant genes incorporatedinto the genome of said engineered cyanobacterium.
 9. The method ofclaim 1 wherein said enzymes are encoded by genes which are present inmultiple copies in said engineered cyanobacterium.
 10. The method ofclaim 1 wherein said enzymes are encoded by genes which are part of anoperon, and wherein the expression of said genes is controlled by asingle promoter.